JP7360174B2 - Activated chimeric receptors and their use in natural killer cell immunotherapy - Google Patents

Description

Related Applications This application is filed in US Provisional Patent Application No. 62/628,788, filed on February 9, 2018, and US Provisional Patent Application No. 62/736,879, filed on September 26, 2018. Claim the benefit of the statement. The entirety of each of these applications is incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILES This application incorporates by reference the Sequence Listing contained in the following ASCII text files, filed contemporaneously with this specification.
a) File name: 44591147002SeqList. txt; Created on February 6, 2019 with a size of 86.4KB

The emergence and persistence of many diseases is characterized by an inadequate immune response against abnormal cells, including malignant and virus-infected cells. Immunotherapy is the use and manipulation of a patient's immune system to treat various diseases.

Immunotherapy represents a new technological advance in the treatment of disease, in which immune cells are engineered to express specific targeting and/or effector molecules that specifically identify and respond to diseased or damaged cells. Ru. This represents a promising advance based, at least in part, on the ability to specifically target diseased or damaged cells, as opposed to more traditional approaches such as chemotherapy, where all cells are affected; The desired result is also that enough healthy cells survive to allow the patient to survive. One immunotherapeutic approach is recombinant expression to activate chimeric receptors on immune cells to achieve targeted recognition and destruction of abnormal cells of interest.

To address this need for specific targeting and destruction, disabling or especially inactivation of diseased or infected cells, chimeras conferring enhanced targeting and cytotoxicity to cells, such as natural killer cells. Provided herein are polynucleotides, amino acids and vectors encoding activating receptors. Further provided are methods for making the cells and using the cells to target and destroy diseased or damaged cells. In some embodiments, polynucleotides encoding activated chimeric receptors that include an extracellular receptor domain and an effector domain that includes a transmembrane region and an intracellular signaling domain are provided, wherein the cellular receptor domain is Contains a peptide that binds with high affinity to the immunizing antigen.

For example, in some embodiments, an activated chimeric receptor comprising an extracellular receptor domain comprising an engineered variant of natural killer group 2 member C (NKG2C) that has a higher affinity for immune ligands of NKG2C. Polynucleotides encoding are provided. In some embodiments, the polynucleotide is such that the encoded NKG2C variant has enhanced binding affinity for an immune ligand, such as an HLA-E/peptide complex, compared to native NKG2C. While designed, unmodified NKG2C has lower binding affinity for HLA-E/peptide complexes. In some embodiments, a modified NKG2C variant is used in the absence of a native or modified NKG2A variant. In some embodiments, full length or other naturally occurring truncated forms of NKG2C are not used.

In some embodiments, natural killer group 2 member A ( Provided are polynucleotides encoding activated chimeric receptors comprising an extracellular receptor domain comprising a fragment of NKG2A). In some embodiments, a modified NKG2A variant is used in the absence of a native or modified NKG2C variant. In some embodiments, full length or other naturally occurring truncated forms of NKG2A are not used.

In some embodiments, the activated chimeric receptor encoded by the polynucleotide includes an effector domain that includes a transmembrane region and an intracellular signaling domain, which domains contain an immunizing antigen of the chimeric receptor, e.g., an HLA - After binding (with enhanced affinity) to the E/peptide complex, it serves to transmit activation and/or costimulatory signals. In some embodiments, the extracellular receptor domain comprises an engineered NKG2C variant conjugated to an effector domain that includes a transmembrane region and an intracellular signaling domain. In some embodiments, the extracellular domain of the engineered NKG2C variant is conjugated to a native NKG2C transmembrane region and/or a native NKG2C intracellular signaling domain. In some embodiments, the modified NKG2C variant comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the modified NKG2C variant is encoded by the nucleic acid sequence of SEQ ID NO: 63, or a fragment thereof. In some embodiments, the NKG2C variant has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about They have 99% homology.

In some embodiments, the activated chimeric receptor comprises a fragment of NKG2A conjugated to an effector domain that includes a transmembrane region and an intracellular signaling domain. In some embodiments, the NKG2A fragment is conjugated to the transmembrane region of native NKG2A and/or the intracellular signaling domain of native NKG2A. In some embodiments, the NKG2A fragment is conjugated to the transmembrane region of native NKG2C and/or the intracellular signaling domain of native NKG2C. In some embodiments, the NKG2A fragment has the sequence of SEQ ID NO: 65. In some embodiments, the fragment of NKG2A is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about They have 99% homology. In one embodiment, the NKG2A fragment is conjugated to an effector domain comprising the CD8a transmembrane domain and one or more of 4-1BB and CD3ζ. In some embodiments, such activated chimeric receptor constructs are encoded by the nucleic acid sequence of SEQ ID NO: 61 or include the amino acid sequence of SEQ ID NO: 62.

In some embodiments, other signaling moieties are used within the effector domain. For example, in some embodiments, the polynucleotide further encodes DNAX activating protein 12 (DAP12) or DAP10. However, in some embodiments, DAP10 is not included in the activating receptor construct and/or not included in another modified construct expressed by the cell. Similarly, in some embodiments, DAP12 is not included in the activating receptor construct and/or in another modified construct expressed by the cell. In some embodiments, the polynucleotide encodes native CD94 or chimeric CD94. In some embodiments, the chimeric CD94 comprises a fragment of CD94 that includes an extracellular receptor domain and an effector domain that includes a transmembrane region and an intracellular signaling domain. In a further embodiment, the chimeric CD94 receptor comprises a fragment of CD94 conjugated to a CD8a transmembrane domain and an effector domain comprising one or more of 4-1BB and CD3ζ. In some embodiments, the chimeric CD94 is encoded by the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, the chimeric CD94 comprises the amino acid sequence of SEQ ID NO:60. In some embodiments, variants, fragments, or truncations of CD94 are also used. For example, in some embodiments, at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% compared to SEQ ID NO: 59 or 60 % homology is used. In one embodiment, the fragment of CD94 has at least 80% homology to SEQ ID NO: 67. In one embodiment, the fragment of CD94 has at least 80% homology to SEQ ID NO: 68.

In some embodiments, the effector domain includes one or more of CD16, NCR1, NCR2, NCR3, 4-1BB, NKp80, DAP10, CD3ζ, 2B4. According to some embodiments, active signaling fragments of any of these signaling moieties are also useful.

In some embodiments, the activated chimeric receptor further comprises a linker and/or hinge region. In some embodiments, these regions (if included) are part of the molecule, e.g., to reduce steric hindrance of the receptor and/or to reduce or eliminate functional challenges that may arise during the formation of tertiary structure. It can function to space other parts. In some embodiments, the activated chimeric receptor comprises a GS linker that is optionally repeatable two or more times. For example, in some embodiments, the GS linker is repeated 3, 4, 5, 6 or more times. In some embodiments, the hinge region includes a glycine-serine repeat motif having the amino acid sequence of SEQ ID NO:31. The repeats need not be consecutive in a "head-to-tail" fashion, but may be spaced from each other. In some embodiments, the chimeric receptor includes a hinge region. In some embodiments, the hinge encoded by the nucleic acid sequence of SEQ ID NO: 5 is used. In a further embodiment, a hinge region encoded by a fragment of the nucleic acid sequence of SEQ ID NO: 5 is used. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, a portion of a beta-adrenergic receptor is used as the hinge region. In some embodiments, the hinge region encoded by the nucleic acid sequence of SEQ ID NO: 40 or SEQ ID NO: 42 is used.

In some embodiments, a signal peptide is also provided. Thus, in some embodiments, the extracellular receptor domain further comprises a CD8a signal peptide comprising the nucleic acid sequence of SEQ ID NO:4.

To facilitate, and in some embodiments enhance, activation signaling by the disclosed chimeric receptors, effector domains are formed to include one or more hemi-ITAM sequences. In some embodiments, the hemi-ITAM comprises either the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of SEQ ID NO: 37. In some embodiments, if more than one sequence of hemi-ITAM (or other transmembrane or signaling component) is provided, the final construct encoded by the polynucleotide will contain a mixture of the sequences (e.g. Heterotandem signaling domains) may also be used. In some embodiments, the effector domain includes one or more ITSM sequences. For example, in some embodiments, the ITSM comprises the amino acid sequence of SEQ ID NO: 15 and/or the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the polynucleotide comprises an extracellular receptor domain that binds to the natural ligand of natural killer group 2 member D (NKG2D), as well as signaling to cells expressing the chimeric receptor upon binding of the NKG2D ligand. It further encodes a chimeric receptor that includes an effector domain that includes a transmembrane region and an intracellular signaling domain that serves for the purpose of transduction. In some embodiments, the chimeric receptor does not use full-length or wild-type NKG2D, but instead uses a fragment of NKG2D that retains the ability to bind one or more ligands of NKG2D. In some embodiments, the chimeric receptor that binds NKG2D is provided on a separate polynucleotide from the polynucleotide encoding the activating receptor (but ultimately both are provided in a single cell). (can be co-expressed).

In some embodiments, the polynucleotide also encodes a short hairpin RNA (shRNA) that specifically inhibits transcription or translation of native NKG2A. In such embodiments, shRNAs that inhibit transcription or translation of native NKG2A, if not removed, result in decreased expression of NKG2A on cells expressing the polynucleotide. In some embodiments, this reduces competition between the higher affinity NKG2A and NKG2C for the immunizing antigen. As a result, NKG2C receptors with relatively lower affinity are able to bind immune antigens (such as HLA-E/peptide complexes) and transmit activation signals to cells (such as NK cells). In one embodiment, the shRNA comprises a nucleotide sequence that hybridizes under stringent conditions to the native NKG2A gene and is substantially identical to a target sequence in the NKG2A gene that is absent from the NKG2A fragment. a sense fragment, and an antisense fragment, where the sense and antisense fragments are separated by a loop fragment. Other mechanisms for reducing expression of the native NKG2A receptor can also be used depending on the embodiment. For example, any other vector containing an antisense sequence capable of disrupting the production of the native NKG2A receptor (but leaving the production of a modified NKG2A fragment/construct as disclosed herein) can be used. . Gene editing techniques can also be used to selectively edit one or more regions of DNA encoding the native NKG2A receptor. Immunology or mechanical separation techniques (affinity immobilization) can also be used to selectively deplete cell populations, such as immune cells such as NK or T cells, of cells expressing the NKG2A receptor.

In some embodiments, a provided polynucleotide encodes or is coexpressed with an additional construct encoding membrane-bound interleukin 15 (mbIL15). Membrane-bound IL15, in some embodiments, promotes proliferation of cells expressing activated chimeric receptors disclosed herein.

In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expression of an activated chimeric receptor.

In addition to the polynucleotide, vectors comprising the polynucleotide are provided herein that deliver and direct the expression of the protein encoded by the polynucleotide in cells, such as immune cells (e.g., NK cells). Formed to facilitate. In some embodiments, the vector is a retrovirus, such as a lentivirus or HIV. Further embodiments are provided with other vectors, such as adenoviruses, adeno-associated viruses, as well as non-viral vectors (eg, liposomes).

Additionally, provided herein are genetically modified cells, such as immune cells, that include a polynucleotide disclosed herein and that express an activated chimeric receptor. A variety of immune cells are used depending on the embodiment. In some embodiments, NK cells are used. In some embodiments, autologous cells (eg, NK cells) modified to express activated chimeric receptors are provided. In further embodiments, allogeneic cells (eg, NK cells) modified to express activated chimeric receptors disclosed herein are provided. In some embodiments, the cell population is enriched for high affinity activated chimeric receptors. In one embodiment, enrichment comprises converting an inhibitory NKG2A receptor to an activating NKG2A receptor (eg, by expressing a polynucleotide disclosed herein). Additionally, this conversion can be coupled with the expression of modified NKG2C receptors that have enhanced affinity for ligands that generate activation signals that transmit NKG2C. Additionally, cell populations can be formed devoid of surface expression of native NKG2A, either by genetic modulation of nucleic acids encoding native NKG2A and/or selective depletion of cells expressing native NKG2A from the population.

Additionally, genetically modified immunization comprising an activating chimeric receptor that is either a modified NKG2C receptor with enhanced affinity for the antigen or an NKG2A variant that has been converted from an inhibitory receptor to an activating receptor. Provided herein are cells, such as NK cells. In some embodiments, such cells also bind to the natural ligand of natural killer group 2 member D (NKG2D) through an extracellular receptor domain (and an effector domain that includes a transmembrane region and an intracellular signaling domain). a polynucleotide encoding a chimeric receptor formed to or a combination thereof.

Further provided herein is a method for enhancing the cytotoxicity of an immune cell in a mammal by administering the immune cell to said mammal, wherein said immune cell is enhanced against an antigen. Expressing an activating chimeric receptor that is either a modified NKG2C receptor or an NKG2A mutant that has been converted from an inhibitory receptor to an activating receptor with a specific affinity. A variety of immune cells are used depending on the embodiment. In some embodiments, NK cells are used. In some embodiments, autologous cells (eg, NK cells) modified to express activated chimeric receptors are provided. In further embodiments, allogeneic cells (eg, NK cells) modified to express activated chimeric receptors disclosed herein are provided. In some embodiments, the method further comprises depleting the population of cells to be administered to the subject of cells expressing native NKG2A. In some embodiments, depletion results in a 20%, 25%, 30%, 40%, or 50% (or more) decrease in cells expressing native NKG2A. In some embodiments, enhanced immune cell-based cytotoxicity is used in treating or preventing cancer or infectious diseases.

Further provided herein is the use of a polynucleotide encoding an activated chimeric receptor in the manufacture of a cell-based medicament to enhance the cytotoxicity of natural killer (NK) cells. As discussed herein, in making a drug, an activated chimeric receptor is a modified NKG2C receptor that has enhanced affinity for the antigen or is converted from an inhibitory receptor to an activating receptor. It may be an NKG2A mutant. In some embodiments, both modified NKG2C and NKG2A variants are used in the manufacture of a medicament for the treatment of cancer or infectious disease.

Although the compositions and related methods outlined above and presented in further detail below describe certain actions taken by a practitioner, it is understood that they may also include direction of those actions by another party. Should. Thus, an act such as "administering a population of NK cells expressing an activated chimeric receptor" includes "directing the administration of a population of NK cells expressing an activated chimeric receptor."

represents a modified receptor according to some embodiments disclosed herein. FIG. 1A shows the HLA- Figure 2 depicts a schematic diagram showing a modified Natural Killer Group 2C (NKG2C) variant (165-168SIIS) engineered for high affinity for E/peptide complexes. FIG. 1B shows NKG2C(165-168SIIS) inserted into an MSCV retroviral vector with green fluorescent protein (GFP) after an internal ribosome entry site (IRES), according to some embodiments disclosed herein. , Dap12, and CD94. Flow cytometry data regarding expression of high affinity activated D12 (SIIS)2C94 receptor complex in purified NKG2C(+)NKG2A(−) NK cells is presented. The expression characteristics of D12(SIIS)2C94 in NK cells transduced with a vector containing only GFP (FIGS. 2A to 2C) and NK cells transduced with a vector containing D12(SIIS)2C94 (FIGS. 2D to 2F) are shown. be done. Figure 2 depicts the design and production of HLA-E molecules with HLA-G signal peptides. Figure 3A depicts the replacement of HLA-E with the HLA-G signal peptide (referred to as GpHLA-E; HLA-E with HLA-G signal peptide). FIG. 3B represents flow cytometry data for exogenous expression of GpHLA-E in solid tumor cell lines HT29, U2OS, ES8, and EW8. Purified NKG2C transduced with D12(SIIS)2C94 against recombinant tumor cell lines HT29-GpHLA-E (Figure 4A), U2OS-GpHLA-E (Figure 4B), and SKBR3-GpHLA-E (Figure 4C). Data are presented for a 4-hour cytotoxicity assay of (+) NKG2A (-) NK cells at the indicated E:T ratios. Data regarding the characterization of transduced NK cells are presented. Figure 5A represents flow cytometry data for NKG2A depletion of NK cells to generate a population of NKG2A(-) NK cells. Figure 5B shows mock-transduced NKG2A (-) against genetically engineered tumor cell lines (i) HT29-GpHLA-E and (ii) U2OS-GpHLA-E incubated in the presence or absence of anti-NKG2A antibody Z199. Data are presented for cytotoxicity assays of NK cell populations or D12(SIIS)2C94-transduced NKG2A(-) NK cells at the indicated E:T ratios. 1 depicts a schematic diagram of an embodiment of a chimeric receptor as disclosed herein. FIG. 6A shows deletion of the N-terminal portion of the transmembrane and inhibitory cytoplasmic domains, as well as the transmembrane domain of CD8a and the cytoplasmic domains of 4-1BB (CD137) and CD3ζ, according to some embodiments disclosed herein. Figure 2 depicts a schematic diagram showing a truncated form of natural killer group 2 member A (NKG2A) and CD94 in which there is a substitution with . The activated NKG2A receptor and the chimeric CD94 receptor form an activated complex (referred to herein as "2A/94BBz") according to some embodiments disclosed herein. FIG. 6B shows chimeric CD94 and activated NKG2A inserted into an MSCV retroviral vector with green fluorescent protein (GFP) after an internal ribosome entry site (IRES), according to some embodiments disclosed herein. Figure 2 depicts a schematic diagram of a construct containing a receptor. Flow cytometry data for expression of activated NKG2A/CD94-41BB-CD3z receptors in transduced NKG2A-depleted NK cells (bottom row) and mock-transduced NK cell populations (top row) are presented. (a) Mock-transduced NK cell populations or expanded cells expressing activated NKG2A/CD94-41BB-CD3z receptors for U2OS-GpHLA-E cells (Figure 8A) and HT29-GpHLA-E cells (Figure 8B). Data are presented for a 4-hour cytotoxicity assay of NK cells at the indicated E:T ratios. Non-limiting embodiments of constructs and portions thereof are provided in accordance with some embodiments of the invention. 3 depicts data regarding the antitumor activity of colorectal adenocarcinoma xenograft models and NK cell constructs according to some embodiments disclosed herein. FIG. 3 depicts survival data from a xenograft model of colorectal adenocarcinoma following administration of NK cell constructs in accordance with some embodiments disclosed herein.

SUMMARY The emergence and persistence of abnormal cells (including viral infections and malignant cells), which underlie many diseases, is enabled by an inadequate immune response against the abnormal cells. The goal of immunotherapy is to initiate or enhance the response of a patient's immune system, such as the ability of immune cells, such as natural killer (NK) cells, to damage, kill, or otherwise inhibit damaged or abnormal cells. It's about supporting them. One immunotherapeutic approach is the recombinant expression of activated chimeric receptors in immune cells to target the recognition and destruction of abnormal cells. Generally, a chimeric receptor comprises an extracellular receptor domain that recognizes a ligand on a target cell, an anchoring transmembrane domain, and an effector domain that transmits activation of a signal upon binding of the ligand. In some embodiments disclosed herein, activated chimeric receptors having the general structure or having variations in the general structure are used. Additionally, in some embodiments, the transmembrane domain and effector domain are separate peptides fused together. In some other embodiments, the transmembrane and effector domains are derived from the same peptide. In some such embodiments, the transmembrane and effector domains include a single peptide (eg, one peptide that, in addition to transmembrane, is balanced and initiates a signal cascade). As discussed in more detail below, the desired degree of expression in immune cells (e.g., NK cells) balances the degree of target binding activity that avoids deleterious effects on non-target cells to induce cytotoxic activity from NK cells. Truncations, mutations, additional linker/spacer elements, dimers, etc. are used to generate activated chimeric receptor constructs. Recombinant expression of activated chimeric receptors as disclosed herein on the surface of immune cells redirects the targeting of the immune cells to the abnormal cells of interest and reduces immune activation upon association. It may be strengthened.

NK Cells for Immunotherapy One immunotherapy approach involves administering to a patient T cells engineered to express activated chimeric receptors and eliciting a positive immune response. However, a drawback of this approach is that it requires the use of autologous cells to prevent the induction of graft-versus-host disease in the patient. As provided in some embodiments disclosed herein, compositions comprising engineered NK cells enjoy several advantages. For example, either autologous or donor-derived allogeneic cells can be used in NK cell approaches. In addition, according to some embodiments, engineered NK cells as provided herein do not exhibit significantly enhanced cytotoxicity toward normal cells. Furthermore, NK cells have significant cytotoxic effects when activated. In view of this, engineered NK cells as provided herein provide an even more effective means of selectively killing diseased target cells by further enhancing their cytotoxic effects. What it can offer is unexpected. Accordingly, in some embodiments, a method of treating or preventing cancer or an infectious disease comprises administering a therapeutically effective amount of a NK cell expressing an activated chimeric receptor described herein. A method is provided. In one embodiment, the NK cells administered are autologous cells. In further embodiments, the NK cells administered are donor-derived (allogeneic) cells.

In some embodiments, association and activation of recombinant NK cells expressing activated chimeric receptors (e.g., by binding to a ligand on target cells) results in cytolytic stress and/or abnormal cells ( direct killing of tumor cells, virus-infected cells, etc.). Accordingly, in some embodiments, a method of enhancing NK cell cytotoxicity comprises administering NK cells engineered to express an activated chimeric receptor described herein. A method is provided. In one embodiment, the NK cells administered are autologous cells. In further embodiments, the NK cells are donor-derived (allogeneic) cells. In some embodiments, engineered NK cells result in indirect destruction or inhibition of stressed and/or abnormal cells (eg, tumor cells, virus-infected cells, etc.).

Activated Chimeric Receptors that Bind HLA-E/Peptide Complexes As noted above, in some embodiments, NK cells recognize and destroy abnormal cells, including tumor cells and virus-infected cells. The cytotoxic activity of these natural immune cells is regulated by the balance of signaling from each of the inhibitory and activating receptors present on the cell surface. The former binds to "self" molecules expressed on the surface of healthy cells, while the latter binds to ligands expressed on abnormal cells. The underlying logic is that the inhibitory receptors on NK cells that interact with "self" molecules are not activated, thereby sparing NK cells from destroying cells with "self" molecules. . In contrast, when NK cells expressing activating receptors interact with "non-self" ligands, for example on abnormal cells, they become activated and a cytotoxic effect follows. Therefore, increased association of activating receptors compared to inhibitory receptors leads to NK cell activation and target cell lysis.

Natural killer group 2 members A (NKG2A) and C (NKG2C) are C-type lectin receptors expressed primarily on the surface of NK cell receptors that influence the balance of inhibitory and activatory signaling. Both NKG2A and NKG2C are capable of forming heterodimers with, for example, the integral membrane glycoprotein CD94. Furthermore, both NKG2C/CD94 and NKG2A/CD94 bind to HLA-E/peptide complexes of non-classical MHC class I molecules in humans. HLA-E plays a special role in cell recognition by NK cells because HLA-E binds to a restricted subset of peptides derived from the signal peptide of classical MHC class I molecules, namely HLA-A, B, C, and G. It has a role. Therefore, NK cells can indirectly monitor the expression of classical MHC class I molecules through the interaction of NKG2C/CD94 and NKG2A/CD94 with HLA-E. NKG2C is an activated receptor, and binding of the HLA-E/peptide complex allows interaction between NKG2C/CD94 and the ITAM-bearing adapter protein DAP12. In contrast, NKG2A is an inhibitory receptor, and NKG2A/CD94 transmits inhibitory signals through the ITIM motif within its cytoplasmic domain.

The ability of NK cells to recognize and destroy abnormal cells, including tumor cells and/or virus-infected cells, makes them potentially useful components of immunotherapeutic approaches (including chimeric receptor-based immunotherapeutic approaches). However, complicating the use of NK cells, HLA-E is upregulated in multiple tumors, and exposure of tumor cells to IFNγ (secreted by immune cells) enhances HLA-E expression. The fact is that Since NKG2C has a lower affinity for peptide complexes compared to NKG2A, increased expression of HLA-E on target cells may function simply as "camouflage". This is a consequence that NKG2A, which is inhibitory even with increased affinity, can outcompete NKG2C for binding to target peptides, thus causing an overall inhibitory effect on NK cell activity.

One approach to address this, as provided in some embodiments, is a polynucleotide encoding an activated chimeric receptor that binds to the HLA-E/peptide complex with enhanced affinity. . In some embodiments, the activated chimeric receptor is an NKG2C mutant that has been modified for enhanced affinity (e.g., compared to unmodified NKG2C affinity) for HLA-E/peptide complexes. Including the body. In some embodiments of the NKG2C variant, residues 165-168 of wild-type NKG2C are replaced with the corresponding residues of NKG2A (SIIS). In some embodiments, the NKG2C variant comprises the amino acid sequence of SEQ ID NO: 58 (SIIS), where SIIS refers to serine-isoleucine-isoleucine-serine. In such embodiments, the affinity of the original NKG2A receptor is "grafted" such that the previously lower affinity NKG2C receptor is modified to have enhanced affinity. In some embodiments, the affinity of mutant NKG2C (as compared to non-mutated NKG2C) is about 20%, about 30%, about 40%, about 50%, or more, depending on the embodiment. To increase.

In some embodiments disclosed herein, polynucleotides encoding activated chimeric receptors, e.g., mutant NKG2C, are provided, wherein the extracellular receptor domain extends from its native transmembrane and/or cellular A fragment of an NKG2C variant that lacks the endodomain, which in some embodiments provides the variant with additional enhanced affinity for HLA-E/peptide complexes. In some embodiments, the NKG2C variant fragment is encoded by SEQ ID NO: 63. In some embodiments, the fragment of the NKG2C variant is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to full-length wild-type NKG2C. . In some embodiments, the fragment may have one or more additional mutations (e.g., insertions, deletions, and/or conservative or non-conservative substitutions) from SEQ ID NO: 63. retains, or in some embodiments enhances, ligand binding function. In some embodiments, NKG2C variant fragments are provided as dimeric, trimeric, or other concatemeric forms; such embodiments provide enhanced ligand binding activity. In some embodiments, the sequence encoding the NKG2C variant fragment is optionally fully or partially codon optimized.

Additionally, in some embodiments a signal peptide is used. The species or sequence of the signal peptide may vary depending on the construct. However, in some embodiments, a signal peptide derived from CD8 is used. In one embodiment, the signal peptide is derived from CD8a and has the sequence of SEQ ID NO:4. In some embodiments, the polynucleotide encodes a NKG2C variant receptor and DAP12. In some embodiments, the polynucleotide encodes a NKG2C variant receptor and CD94. In some embodiments, the polynucleotide encodes the NKG2C variant receptor, CD94, and DAP12. In some embodiments, where NKG2C is used, the cell is further modified to alter the expression of native NKG2A (eg, reduce or eliminate transcription and/or reduce or eliminate translation).

In some embodiments, NKG2A is modified to deliver an activating signal upon binding of the HLA-E/peptide complex, as opposed to its normal delivery of an inhibitory signal. As used herein, "NKG2A" refers to any variant, derivative, or isoform of the NKG2A gene or encoded protein, including but not limited to NKG2B. Thus, according to some embodiments disclosed herein, polynucleotides encoding activated chimeric receptors are provided, wherein the extracellular receptor domain is devoid of its native transmembrane or intracellular domains. but advantageously a fragment of NKG2A that retains its ability to bind to the HLA-E/peptide complex. Thus, in some embodiments, a chimeric receptor encoded by a polypeptide disclosed herein does not include an ITIM, an ITAM, or a hemi-ITAM/hemi-ITAM. In some embodiments, the NKG2A fragment is encoded by SEQ ID NO: 65. In some embodiments, the fragment of NKG2A is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to full-length wild-type NKG2A. In some embodiments, the fragment may have one or more additional mutations (e.g., insertions, deletions, and/or conservative or non-conservative substitutions) from SEQ ID NO: 65. retains, or in some embodiments enhances, ligand binding function. In some embodiments, the NKG2A fragment is provided as a dimeric, trimeric, or other concatemeric form; such embodiments provide enhanced ligand binding activity. In some embodiments, the sequence encoding the NKG2A fragment is optionally fully or partially codon optimized. Additionally, in some embodiments a signal peptide is used. The species or sequence of the signal peptide may vary depending on the construct. However, in some embodiments, a signal peptide derived from CD8 is used. In one embodiment, the signal peptide is derived from CD8a and has the sequence of SEQ ID NO:4. In some embodiments, the polynucleotide encodes an activated NKG2A receptor and a CD94 receptor.

In some embodiments, CD94 is modified to deliver an activation signal upon binding of the HLA-E/peptide complex. Thus, according to some embodiments disclosed herein, polynucleotides encoding activated chimeric receptors are provided, wherein the extracellular receptor domain is devoid of its native transmembrane or intracellular domains. but advantageously a fragment of CD94 that retains its ability to dimerize with NKG2A and NKG2C. In some embodiments, the CD94 fragment is encoded by SEQ ID NO: 67. In some embodiments, the fragment of CD94 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to full-length wild-type CD94. In some embodiments, the fragment may have one or more additional mutations (e.g., insertions, deletions, and/or conservative or non-conservative substitutions) from SEQ ID NO: 67. retains, or in some embodiments enhances, ligand binding function. In some embodiments, the CD94 fragment comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the fragment of CD94 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to full-length wild-type CD94. In some embodiments, the fragment may have one or more additional mutations (e.g., insertions, deletions, and/or conservative or non-conservative substitutions) from SEQ ID NO: 68. retains, or in some embodiments enhances, ligand binding function. In some embodiments, CD94 fragments are provided as dimeric, trimeric, or other concatemeric forms; such embodiments provide enhanced ligand binding activity. In some embodiments, the sequence encoding the CD94 fragment is optionally fully or partially codon optimized. Additionally, in some embodiments a signal peptide is used. The species or sequence of the signal peptide may vary depending on the construct. However, in some embodiments, a signal peptide derived from CD8 is used. In one embodiment, the signal peptide is derived from CD8a and has the sequence of SEQ ID NO:4. In some embodiments, the polynucleotide encodes a chimeric CD94 receptor and an activated NKG2A receptor. In some embodiments, the polynucleotide encodes an activated NKG2C variant receptor and a chimeric CD94 receptor.

In some embodiments, a polynucleotide encoding an activating chimeric receptor disclosed herein is an NKG2A-depleted cell population (e.g., depleted or substantially depleted of cells expressing NKG2A; or expressed in a completely depleted population of NK cells - eg, immunodepletion.

Transmembrane, Signal Transduction and Combination Domains As noted above, a typical activated chimeric receptor structure includes at least one transmembrane domain that connects the ligand binding domain to the signal transduction domain. In some embodiments, however, the transmembrane domain may also serve to provide a signaling function.

In some embodiments, the CD94 fragment, NKG2A fragment, and/or NKG2C variant retains at least a portion of its normal transmembrane domain. In some embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein that is normally expressed on both T cells and NK cells. In some embodiments, the transmembrane domain comprises CD8α, while in some embodiments CD8β is used. In some embodiments, the "hinge" of CD8α (eg, the portion between the extracellular and intracellular domains) has the sequence of SEQ ID NO: 5. In some embodiments, the CD8α is truncated or modified such that it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD8α having the sequence of SEQ ID NO: 5. can be done. In some embodiments, CD8β has the sequence of SEQ ID NO: 6. In some embodiments, the CD8β is truncated or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD8β having the sequence of SEQ ID NO: 6. can be done. In some embodiments, dimers or triplets or repeated sequences of CD8α and CD8β are used.

In some embodiments, the transmembrane domain includes CD16, which also serves as a signal transduction domain. CD16 exists in two isoforms a and b (also known as Fcγ receptors IIIa and IIIb, respectively). These receptors normally bind to the Fc portion of IgG antibodies, which in turn activates NK cells. Thus, in some embodiments, the transmembrane domain comprises CD16a, while in some embodiments CD16b is used. In some embodiments, CD16a has the sequence SEQ ID NO:7. In some embodiments, the CD16a is truncated or modified such that it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD16a having the sequence of SEQ ID NO: 7. can be done. In some embodiments, CD16b has the sequence SEQ ID NO:8. In some embodiments, the CD16b is truncated or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD16b having the sequence of SEQ ID NO: 8. can be done. In some embodiments, a dimer of CD16a and CD16b is used. In some embodiments, modifications to the CD16 transmembrane domain include additional nucleic acid residues to increase the length of the domain. Alternatively, CD16 may be shortened. Modifications to the length of CD16 may advantageously promote enhanced ligand-receptor interaction.

In some embodiments, the activated chimeric receptor herein comprises the natural killer receptor 2B4 domain (referred to herein as "2B4" and also known as CD244), which also serves as a signaling domain. ). 2B4 is expressed on NK cells and controls non-major histocompatibility complex (MHC)-restricted killing through the interaction of this receptor with its ligand on target cells. In some embodiments, the transmembrane domain comprises 2B4, while in some embodiments the 2B4 domain is employed as an intracellular signaling domain. In some embodiments, 2B4 has the sequence SEQ ID NO:9. In some embodiments, 2B4 is truncated or modified such that it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to 2B4 having the sequence of SEQ ID NO: 9. can be done. In some embodiments, 2B4 is used as the only transmembrane/signaling domain in the construct, although in some embodiments 2B4 can be used in combination with one or more other domains. For example, in some embodiments a combination of CD16, 4-1BB and/or 2B4 is used.

In some embodiments, signaling is accomplished through DAP10. In some embodiments, a dimer of DAP10 is used. In some embodiments, the transmembrane domain comprises DAP10. In some embodiments, DAP10 has the sequence SEQ ID NO:10. In some embodiments, DAP10 is truncated or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to DAP10 having the sequence of SEQ ID NO: 10. can be done. Similarly, in some embodiments, DAP12 can also be used to transduce such signals. In some embodiments, DAP12 has the sequence SEQ ID NO:11. In some embodiments, DAP12 is truncated or modified such that it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to DAP12 having the sequence of SEQ ID NO: 11. can be done. In some embodiments, a heterodimer of DAP10 and DAP12 is used. However, in some embodiments, neither DAP10 nor DAP12 is used in either the NKG2A, NKG2C, or NKG2D constructs.

In some embodiments, signaling is provided through 4-1BB (also known as CD137 and tumor necrosis factor receptor superfamily member 9 (TNFRSF9)). 4-1BB is a costimulatory immune checkpoint that typically functions as a stimulatory molecule for activated immune cells (e.g., cross-linking of 4-1BB enhances T cell proliferation and cytolytic activity). It is a molecule. However, in some embodiments, the functions of 4-1BB are advantageously used in conjunction with NK cells. In some embodiments, 4-1BB has the sequence of SEQ ID NO:12. In some embodiments, 4-1BB is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to 4-1BB having the sequence of SEQ ID NO: 12. It can be truncated or modified. In some embodiments, 4-1BB is the only signaling domain, but as discussed above, in some embodiments 4-1BB is the only signaling domain disclosed herein. /signaling domains. For example, in some embodiments, CD16 provides a synergistic stimulatory effect in conjunction with 4-1BB, resulting in particularly effective (eg, cytotoxic) NK cells. In some embodiments, DAP10 provides a synergistic stimulatory effect in conjunction with 4-1BB, resulting in particularly effective (eg, cytotoxic) NK cells. In some embodiments, DAP10, in combination with 4-1BB and 2B4, provides a synergistic stimulatory effect, resulting in particularly effective (eg, cytotoxic) NK cells.

In some embodiments, the signaling domain comprises at least a portion of the CD3 T cell receptor complex. The T cell receptor complex contains multiple subunits, including ζ, α, β, γ, δ, and ε subunits. In some embodiments, NK cells engineered according to some embodiments disclosed herein include at least one of these subunits (or fragments thereof). In some embodiments, the signaling domain comprises a CD3ζ subunit. In some embodiments, CD3ζ has the sequence SEQ ID NO: 13. In some embodiments, the CD3ζ is truncated or modified such that it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD3ζ having the sequence of SEQ ID NO: 13. can be done. In some embodiments, CD3ζ is mutated (eg, amino acid mutations, insertions or deletions) such that the domain no longer matches the canonical immunoreceptor tyrosine-based activation motif or ITAM motif. Thus, in some embodiments, the NK cell comprises an engineered receptor that does not contain an ITAM motif. In some embodiments, CD3ζ is not used. In some embodiments, the resulting engineered NK cells exhibit enhanced cytotoxicity, particularly towards target cells, while adverse side effects are limited or reduced. In some embodiments, this is due to synergistic interactions of the various parts of the chimeric receptor used in that given embodiment. In some embodiments, CD3ζ provides a synergistic stimulatory effect in conjunction with 4-1BB, resulting in particularly effective (eg, cytotoxic) NK cells. In some embodiments, CD3ζ provides a synergistic stimulatory effect in conjunction with 2B4, resulting in particularly effective (eg, cytotoxic) NK cells.

In still further embodiments, the signaling portion of the activated chimeric receptor comprises a portion of an ITAM, such as a hemitam. In some embodiments, these portions do not constitute a canonical ITAM sequence, but rather include portions that may further transmit signals required for NK cell cytotoxicity. In some embodiments, the hemitum has the sequence of SEQ ID NO: 14 (where X can be any residue). In some embodiments, the hemitum is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to a hemitum having the sequence of SEQ ID NO: 14. It can be truncated or modified. In some embodiments, the activated chimeric receptor construct comprises the hemitam of SEQ ID NO: 14. In some embodiments, multiple hemitams can be used in a head-to-tail, tail-to-head, head-to-head, or tail-to-tail arrangement, for example. In some embodiments, the presence of at least one hemitum provides enhanced signaling and cytotoxicity to NK cells that include activated chimeric receptors using the at least one hemitam. As discussed in more detail below, some activated chimeric receptors include NKp80, one non-limiting example of a hemitam.

In some embodiments, additional signaling regions are used, including, for example, signaling regions from the signaling lymphocyte activation molecule (SLAM) family of receptors. These receptors include, but are not limited to, 2B4 (discussed above). The SLAM family of receptors share a common tyrosine-based motif in their cytoplasmic tails. The motif is S/TxYxxL/I (SEQ ID NO: 15), termed the immunoreceptor tyrosine-based switch motif (ITSM). These receptors transmit activation signals through the SLAM-associated protein (SAP, encoded by the gene SH2D1A), which recruits the tyrosine kinase Fyn. Thus, according to some embodiments, the signal transduction region includes a polypeptide sequence (or a nucleic acid encoding the same) that includes an ITSM motif. In some embodiments, the ITSM motif need not be fully encoded, but the signaling region can transmit activation signals through SAP (or another similar pathway). In some embodiments, the ITSM motif has the sequence of SEQ ID NO: 15, where X can be any amino acid residue. In some embodiments, the ITSM motif is truncated such that it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the ITSM motif having the sequence of SEQ ID NO: 15. or may be modified. In some embodiments, the ITSM motif comprises the sequence of SEQ ID NO: 15.

In addition to these variations in extracellular receptor domains, transmembrane domains, and signaling domains (and combinations of transmembrane/signaling domains), in some embodiments additional coactivating molecules may be provided. . For example, in some embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, the presence of mbIL15 on NK cells functions to further enhance the cytotoxic effects of NK cells by synergistically increasing NK cell proliferation and longevity. In some embodiments, mbIL15 has the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, mbIL15 can be truncated or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the sequence of SEQ ID NO: 16. In some embodiments, mbIL15 has the amino acid sequence of SEQ ID NO: 17. In conjunction with the activated chimeric receptors disclosed herein, such embodiments provide NK cell compositions that are particularly effective for targeting and destroying specific target cells.

Chimeric receptor constructs can be produced and expressed in NK cells to target and destroy specific target cells (e.g., diseased or cancerous cells) in view of the present disclosure described herein. A variety of activated chimeric receptors exist. Non-limiting examples of such chimeric receptors are discussed in more detail below.

As discussed above, parts of the T cell receptor complex, specifically CD3ζ, function as potent activators of immune signaling cascades. Similarly, receptor 4-1BB, a member of the tumor necrosis factor superfamily, activates NK cells upon binding of ligand. In some embodiments, these two signaling components act synergistically upon binding of the ligand to the chimeric receptor to activate the NK cell. Accordingly, in some embodiments, an NKG2A fragment that binds to an HLA-E/peptide complex includes an extracellular receptor domain of the NKG2A fragment, a CD8 transmembrane region, and an effector domain that includes the signaling domains of 4-1BB and CD3ζ. Polynucleotides encoding a /CD8a/4-1BB/CD3ζ chimeric receptor are provided. In one embodiment, the chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:61. In yet another embodiment, the NKG2A/CD8a/4-1BB/CD3ζ chimeric receptor comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, this construct is particularly effective when the NK cells co-express mbIL15, where mbIL15 provides additional synergistic effects with respect to NK cell activation and cytotoxicity. In some embodiments, the sequence of the chimeric receptor may differ from SEQ ID NO: 61, but depending on the embodiment, the sequence is at least 70%, at least 75%, at least 80%, at least 85%, Maintain at least 90%, or at least 95% homology. In some embodiments, while the chimeric receptor may differ from SEQ ID NO: 61, the chimeric receptor retains NK cell activation and/or cytotoxic function, or in some embodiments We are increasing it.

In addition, in some embodiments, a CD94/CD8a/4-1BB/CD3ζ chimeric receptor comprises a CD94 fragment extracellular receptor domain, a CD8 transmembrane region, and an effector domain that includes 4-1BB and CD3ζ signaling domains. Polynucleotides encoding the body are provided. In one embodiment, the chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO: 59. In yet another embodiment, the CD94/CD8a/4-1BB/CD3ζ chimeric receptor comprises the amino acid sequence of SEQ ID NO:60. In some embodiments, this construct is particularly effective when NK cells co-express mbIL15, where mbIL15 provides additional synergistic effects with respect to long-term activation and cytotoxicity (e.g., in vivo persistence) of NK cells. do. In some embodiments, the sequence of the chimeric receptor may differ from SEQ ID NO: 59, but depending on the embodiment, the sequence is at least 70%, at least 75%, at least 80%, at least 85%, Maintain at least 90%, or at least 95% homology. In some embodiments, while the chimeric receptor may differ from SEQ ID NO: 59, the chimeric receptor retains NK cell activation and/or cytotoxic function, or in some embodiments We are increasing it.

Receptor 2B4 has several immunoreceptor tyrosine-based switch motifs (ITSMs) and has the potential to transmit activation signals. Similarly, signaling through receptor 4-1BB, tumor necrosis factor superfamily members also activate NK cells upon ligand binding. Accordingly, in some embodiments, the ability of these signaling molecules to unexpectedly cooperate to effectively generate cytotoxic NK cells can be exploited to utilize fragments of NKG2A, CD94, or NKG2C variants. Polynucleotides are provided that encode activated chimeric receptors that include an extracellular receptor domain, a CD8a transmembrane region, and an effector domain that includes 4-1BB and 2B4 signaling domains. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15.

In some embodiments, a combination of 2B4 and CD3ζ is used with NK cells to produce enhanced cytotoxicity against target cells. Accordingly, in some embodiments, an activated chimeric receptor comprising an extracellular receptor domain comprising a fragment of NKG2A, CD94, or an NKG2C variant, an effector domain comprising a CD8a transmembrane region, and a signaling domain of CD3ζ and 2B4. Polynucleotides encoding the body are provided. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15. As discussed above, 4-1BB, like CD3ζ and 2B4, is a potent activator of immune signaling cascades. In some embodiments, these three signaling components act in a synergistic manner to activate NK cells upon binding of the ligand to the chimeric receptor.

In some embodiments, an activated chimeric receptor comprising an extracellular receptor domain comprising a fragment of NKG2A, CD94, or an NKG2C variant, a CD8a transmembrane region, and an effector domain comprising the signaling domains of 4-1BB and DAP10. Polynucleotides encoding the body are provided. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15.

In some further embodiments, the transmembrane and effector domains (and related functions) of the activated chimeric receptor are derived from the same peptide. CD16 is a potent activating receptor expressed on the surface of NK cells. Accordingly, in some embodiments, an NKG2A/CD16 chimeric receptor, an NKG2C mutant/ Polynucleotides encoding CD16 chimeric receptors or CD94/CD16 chimeric receptors are provided. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15.

In some further embodiments, polynucleotides encoding NKG2A/CD16/4-1BB chimeric receptors, CD94/CD16/4-1BB chimeric receptors, or NKG2C variant/CD16/4-1BB chimeric receptors are provided. 4-1BB, where the signaling domain of 4-1BB acts as a second transducer of activation signals in the effector domain. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15.

NCR1 (NKp46), NCR2 (NKp44) and NCR3 (NKp30) are receptors on NK cells that transmit activation signals upon ligand binding. Accordingly, in some embodiments, an NKG2A/NCR1 chimeric receptor, a CD94/NCR1 chimeric receptor, comprising both a transmembrane region and an intracellular effector domain, and comprising an NKG2A, CD94, or NKG2C variant fragment, and an NCR1 peptide. Polynucleotides encoding NKG2C mutant/NCR1 chimeric receptors are provided.

In some further embodiments, polynucleotides encoding NKG2A/NCR1/4-1BB chimeric receptors, CD94/NCR1/4-1BB chimeric receptors, or NKG2C variant/NCR1/4-1BB chimeric receptors are provided. 4-1BB, where the signaling domain of 4-1BB acts as a second transducer of activation signals in the effector domain, resulting in synergistically enhanced NK cell activation and cytotoxicity. In some further embodiments, the NKG2A/NCR2 chimeric receptor, the CD94/NCR2 chimeric receptor, the NKG2C variant/NCR2 chimeric receptor, comprises both a transmembrane region and an intracellular effector domain, and comprises an NCR2 peptide. Polynucleotides are provided. Similar to NCR1, in some embodiments these constructs may be particularly suitable for use in generating NK cells expressing chimeric receptors due to their relatively small size and simplicity in sequence. However, in some embodiments, despite the apparent simplicity of the constructs, they retain the ability to generate highly effective NK cells. Additionally, in some embodiments, these constructs can optionally be co-expressed with mbIL15.

In some further embodiments, the NKG2A/NCR3 chimeric receptor, CD94/NCR3 chimeric receptor, or NKG2C variant/NCR3 chimeric receptor comprises both a transmembrane region and an intracellular effector domain and comprises an NCR3 peptide. Encoding polynucleotides are provided. Similar to NCR1 and/or NCR2, in some embodiments these constructs are amenable to use in generating NK cells expressing activated chimeric receptors due to their relatively small size and simplicity in sequence. may be particularly suitable. However, in some embodiments, despite the apparent simplicity of the constructs, they retain the ability to generate highly effective NK cells.

In some further embodiments, polynucleotides encoding NKG2A/NCR2/4-1BB chimeric receptors, CD94/NCR2/4-1BB chimeric receptors, or NKG2C variant/NCR2/4-1BB chimeric receptors are provided. Here, the signaling domain of 4-1BB acts as a second transducer of the activation signal in the effector domain, thereby creating synergy between the signaling domain and cytotoxic NK cells in an unexpectedly effective manner. bring about an effect. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15.

In some further embodiments, polynucleotides encoding NKG2A/NCR3/4-1BB chimeric receptors, CD94/NCR3/4-1BB chimeric receptors, or NKG2C variant/NCR3/4-1BB chimeric receptors are provided. Here, the signaling domain of 4-1BB acts as a second transducer of the activation signal in the effector domain, thereby creating synergy between the signaling domain and cytotoxic NK cells in an unexpectedly effective manner. bring about an effect. Additionally, in some embodiments, this construct can optionally be co-expressed with mbIL15.

In some embodiments, the surface expression and efficacy of the chimeric receptors disclosed herein are determined by the combination of the transmembrane domain and any one of the CD94 fragment, the NKG2A fragment, or the NKG2C fragment, in some embodiments. enhanced by variations in the spacer region (hinge) located within the extracellular domain between. In some embodiments, the hinge region may be included between other portions of the activated chimeric receptor (eg, between an intracellular domain and a transmembrane domain or between multiple intracellular domains). In some embodiments, domains that serve specific purposes disclosed elsewhere herein may serve additional functions. For example, in some embodiments, CD8a is repurposed to function as a hinge region (in some embodiments encoded by the nucleic acid sequence of SEQ ID NO: 5). In yet another embodiment, the hinge region comprises an N-terminal truncated form of CD8a and/or a C-terminal truncated form of CD8a. Depending on the embodiment, these truncations are at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% homologous to the hinge encoded by SEQ ID NO:5. obtain. In some additional embodiments, the hinge includes a span of glycine and serine residues (referred to herein as a "GS linker"), and GSn has the sequence (Gly-Gly-Gly -Gly-Ser)n (SEQ ID NO: 42). In one embodiment, the hinge includes both CD8a and GS3 and is encoded by the amino acid sequence of SEQ ID NO: 32, eg, n=3. In additional embodiments, the value of n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, depending on the embodiment. or higher. In some embodiments, this hinge may also be configured as GSn/CD8a. Alternatively, the GS linker may include the entire hinge region. In one such embodiment, the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 33. In another such embodiment, the hinge region is encoded by the nucleic acid sequence of SEQ ID NO:34.

In some embodiments, the activated chimeric receptor constructs utilize the 2B4 intracellular signaling domain. In some embodiments, this domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the 2B4 domain is encoded by the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the sequence of the 2B4 intracellular domain used in the activated chimeric receptor may differ from SEQ ID NO: 36, but depending on the embodiment, at least 70%, at least 75% from SEQ ID NO: 36. , remain at least 80%, at least 85%, at least 90% or at least 95% homologous. In some embodiments, the signaling domain of the activating chimeric receptor may differ from SEQ ID NO: 36, but the activating chimeric receptor retains NK cell activation and/or cytotoxic function. or, in some embodiments, enhanced. Similarly, in some embodiments, the NKp80 intracellular domain is used in some embodiments. In some embodiments, this NKp80 domain is the only intracellular signaling domain, but in some embodiments, this domain is used in conjunction with one or more additional domains. In some embodiments, the NKp80 is encoded by the amino acid sequence of SEQ ID NO: 37. In some embodiments, the NKp80 domain is encoded by the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the sequence of the NKp80 intracellular domain used in the chimeric receptor can differ from SEQ ID NO: 38, but depending on the embodiment, at least 70%, at least 75%, at least remain 80%, at least 85%, at least 90% or at least 95% homologous. In some embodiments, the signaling domain of the activating chimeric receptor may differ from SEQ ID NO: 38, but the chimeric receptor retains NK cell activation and/or cytotoxic function; or in some embodiments augmented.

In some embodiments, the activated chimeric receptor uses a portion of a beta-adrenergic receptor as the transmembrane domain. In some embodiments, the portion includes a portion of the beta-adrenergic extracellular domain. In some embodiments, the portion is part of the beta-adrenergic receptor transmembrane domain. In some embodiments, a combination of the extracellular and transmembrane domains of the beta-adrenergic receptor is used. Depending on the embodiment, this moiety is derived from β-1 and /β-2 adrenergic receptors. In some embodiments, a portion of the N-terminal extracellular region of the β-2 adrenergic receptor is used. In some embodiments, the portion has the amino acid sequence of SEQ ID NO: 39. In some embodiments, the extracellular β-2 adrenergic domain is encoded by the nucleic acid sequence of SEQ ID NO:40. In some embodiments, the sequence of the extracellular β-2 adrenergic domain used in the chimeric receptor can differ from SEQ ID NO: 39, but depending on the embodiment, the sequence is at least 70%, at least 75% different from SEQ ID NO: 39. %, at least 80%, at least 85%, at least 90% or at least 95% homologous. In some embodiments, the first transmembrane helix of the β-2 adrenergic receptor is optionally used with an extracellular β-2 adrenergic domain. In some embodiments, the first transmembrane helix of the β-2 adrenergic receptor has the amino acid sequence of SEQ ID NO:41. In some embodiments, the first transmembrane helix of the β-2 adrenergic receptor is encoded by the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the sequence of the first transmembrane helix of the β-2 adrenergic receptor used in the chimeric receptor can be different from SEQ ID NO: 41, but depending on the embodiment, remain at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% homologous.

As discussed above, in some embodiments, codon-optimized sequences are used. For example, in some embodiments, codon optimization (full or partial) is performed on extracellular receptor domains that include fragments of CD94, NKG2A, or NKG2C variants. However, in some embodiments, no codon optimization is performed. In some embodiments, a chimeric receptor construct is provided with an extracellular domain comprising a fragment of a non-optimized NKG2A, CD94, or NKG2C variant, a CD8a hinge, and a 4-1BB signaling domain. In some embodiments, the chimeric receptor construct is provided with an extracellular domain comprising a fragment of a non-optimized NKG2A, CD94, or NKG2C variant, a CD8a hinge and transmembrane domain, and a 4-1BB signaling domain. be done.

In some embodiments, an activated chimeric receptor construct is provided with an extracellular domain comprising a fragment of a non-optimized NKG2A, CD94, or NKG2C variant, a CD8a hinge, and an NKp80 signaling domain. In some embodiments, a chimeric receptor construct is provided with an extracellular domain comprising a fragment of a non-optimized NKG2A, CD94, or NKG2C variant, a CD8a hinge and transmembrane domain, and an NKp80 signaling domain. . In some embodiments, a GS linker, such as a GS3 linker, connects 4-1BB and the NKp80 domain.

In some embodiments, the CD8 transmembrane domain is conjugated to the 2B4 intracellular domain. In some embodiments, the CD8 transmembrane domain is replaced with a 2B4 domain that is transmembrane and intracellular. In some embodiments, the CD8 transmembrane domain is replaced with 2B4 and 4-1BB is expressed in a proximal configuration.

In some embodiments, the CD16 intracellular signaling domain is conjugated to a CD3ζ or γ subunit that is exogenously expressed in trans to the activated chimeric receptors described herein. As discussed above, such constructs may unexpectedly result in enhanced signal transduction and therefore an unexpected increase in NK cell cytotoxic effects.

In some embodiments, the activated chimeric receptor is configured to dimerize, as discussed in further detail herein. In some embodiments, a receptor comprising a fragment of CD94, a fragment of NKG2A, or a NKG2C variant according to some embodiments disclosed herein is optionally dimerized. Dimerization may include homodimers or heterodimers depending on the embodiment. In some embodiments, upon dimerization, the avidity of this activated chimeric receptor (and thus the NK cells expressing this receptor) is increased to provide an equivalent balance in reducing (or lack of) deleterious toxic effects. and is shifted to better ligand recognition. In further embodiments, the extracellular receptor domain further comprises a CD8a signal peptide. In some embodiments, the activated chimeric receptor utilizes internal dimers or repeats of one or more component subunits. For example, in some embodiments, the chimeric receptor comprises an NKG2C variant extracellular domain conjugated to a second NKG2C variant extracellular domain and a transmembrane/signaling region (or together with a separate signaling region). separate transmembrane regions). In some embodiments, one or more of the NKG2D extracellular domains are codon optimized. In some embodiments, two NKG2D extracellular domains are separated by a linker (eg, a GSn linker). In one embodiment, a GS3 linker is used. In some embodiments, the transmembrane domain comprises the extracellular region of a beta-adrenergic receptor. In some embodiments, the transmembrane domain The transmembrane domain comprises an extracellular region of a β-2 adrenergic receptor and further comprises a first transmembrane domain of a β-2 adrenergic receptor. In some embodiments, the signaling region comprises 4-1BB. In some embodiments, the signaling region comprises NKp80. In some embodiments, the signaling region comprises the CD16 transmembrane-intracellular domain. In some embodiments, the signaling region comprises 4-1BB with the NKp80 or CD16 transmembrane-intracellular domain.

According to some embodiments disclosed herein, additional chimeric receptors are provided that utilize codon-optimized extracellular receptor domains (optionally, the constructs utilize codon-optimized extracellular receptor domains). (can also be replicated by unoptimized or partially optimized domains). For example, in some embodiments, a codon-optimized extracellular domain is conjugated to a hinge and at least two domains (eg, a transmembrane domain and a signal transduction domain). In some embodiments, multiple signaling domains enhance the cytotoxic efficacy of NK cells because multiple non-redundant signal cascades are initiated. In some embodiments, these multiple pathways may converge on a single signaling molecule (e.g., IFNγ), but due to the overall size of the signaling molecule that drives the cytotoxic endpoint, The overall cytotoxic effect is unexpectedly increased.

In still further embodiments, certain components of the activated chimeric receptor are replaced with one or more additional subunits that provide enhanced efficacy (e.g., NK cell activation or cytotoxicity). obtain. For example, in one embodiment, the CD16 intracellular signaling domain can be replaced with a quadruple repeat of DAP10 (eg, 4xDAP10). In additional embodiments, the CD16 intracellular signaling domain may be replaced with a Zap70 subunit. Certain such embodiments result in unexpected enhancement of NK cell cytotoxicity.

In some additional embodiments, the effector domain comprises one or more consensus hemi-ITAM sequences to enhance activation signaling upon binding of a ligand. In additional embodiments, inclusion of a GS linker between the 4-1BB, CD16, NCR1, NCR2 and/or NCR3 signaling domains enhances signal transduction. Furthermore, in some embodiments, one or both of CD3ζ and FcRγ are further expressed (on the same or separate constructs) with the chimeric receptors described herein, whereby signaling is unexpectedly is also enhanced, thereby unexpectedly increasing the cytotoxic effect of NK cells. Depending on the embodiment, the engineered expression of one or more of CD3ζ and FcRγ complements the endogenous expression of these molecules by NK cells, thereby further enhancing NK cell signaling and ultimate cytotoxic efficacy. be done.

Optionally, depending on the embodiment, any of the polynucleotides disclosed herein may also encode one or more truncations and/or variants of the constituent subunits of the activated chimeric receptor; It may still retain the ability to target NK cells to target cells and, in some embodiments, may unexpectedly enhance cytotoxicity upon binding. In addition, any of the polynucleotides disclosed herein may optionally also include codon-optimized nucleotide sequences encoding the various constituent subunits of the chimeric receptor. As used herein, the terms "fragment" and "truncated" shall be given their normal meanings and shall also include N-terminal deletion variants and C-terminal deletion variants of the protein.

Polynucleotides encoding activated chimeric receptors described herein can be inserted into vectors to achieve recombinant protein expression in NK cells. In one embodiment, the polynucleotide is operably linked to at least one regulatory element for expression of the activated chimeric receptor. In specific embodiments, transcriptional regulatory elements (e.g., internal ribosome entry site (IRES) or enhancer elements) heterologous to the peptides disclosed herein are utilized to regulate the transcription of this activated chimeric receptor. is instructed. Depending on the embodiment, the various components of the activated chimeric receptor can be delivered to NK cells in a single vector or alternatively in multiple vectors. In some embodiments, the activated chimeric receptor construct is delivered in a single vector, but another factor that enhances the efficacy of the activated chimeric receptor (e.g., mbIL15) is delivered in a separate vector. be done. In some embodiments, an activated chimeric receptor and a factor that enhances the efficacy of the activated chimeric receptor (eg, mbIL15) are delivered in a single vector. Regardless of the number of vectors used, any polynucleotide may optionally include a tag sequence, allowing recognition of the presence of NK cells expressing this construct. For example, in some embodiments, the FLAG tag (DYKDDDDK, SEQ ID NO: 55) is used. Also available are other tag sequences, such as the polyhistidine tag (His-tag) (HHHHHH, SEQ ID NO: 56), the HA-tag or the myc-tag (EQKLISEEDL; SEQ ID NO: 57). Instead, green fluorescent protein or other fluorescent moieties are used. Combinations of tag types may also be used to individually recognize subcomponents of a chimeric receptor.

In some embodiments, the polynucleotide encoding the activated chimeric receptor is an mRNA that can be introduced into NK cells by electroporation. In another embodiment, the vector is a virus (preferably a retrovirus) that can be introduced into NK cells by transduction. In some embodiments, the vector is a murine stem cell virus (MSCV). In additional embodiments, other vectors may be used, such as lentiviruses, adenoviruses, adeno-associated viruses, and the like. In some embodiments, non-HIV derived retroviruses are used. The vector chosen will depend on a variety of factors, including, but not limited to, the strength of the transcriptional regulatory elements and the cells used to express the protein. The vector can be a plasmid, phagemid, cosmid, viral vector, phage, artificial chromosome and the like. In additional embodiments, the vector may be an episomal, heterologously or homologously integrating vector, which may be modified by any suitable means for transforming the vector (transformation, gene transfer, , conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc.) into appropriate cells. In some embodiments, other approaches are used to induce expression of chimeric receptors in NK cells, including, for example: the SV40 early promoter region, the 3' long end of the Rous sarcoma virus. Promoters contained in repeats, herpes thymidine kinase promoter, regulatory sequence of metallothionein gene, adenovirus (ADV) promoter, cytomegalovirus (CMV) promoter, bovine papillomavirus (BPV) promoter, parovirus B19p6 promoter, β-lactamase promoter, tac promoter, nopaline synthetase promoter region or cauliflower mosaic virus 35S RNA promoter, promoter of ribulose bisphosphate carboxylase, Gal 4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, myeloproliferative sarcoma virus enhancer, engineered Synthetic MND promoter and alkaline phosphatase promoter containing the U3 region of MoMuLV LTR.

Natural killer cells can be engineered to express the activated chimeric receptors disclosed herein. Activated chimeric receptor expression constructs can be introduced into NK cells using any of the techniques known to those skilled in the art. In one embodiment, the activated chimeric receptor is transiently expressed in NK cells. In another embodiment, the activated chimeric receptor is stably expressed in NK cells. In additional embodiments, the NK cell is an autologous cell. In yet another embodiment, the NK cell is a donor-derived (allogeneic) cell.

Further provided herein is a method of treating a subject with cancer or an infectious disease, the method comprising: treating a subject with cancer or an infectious disease, the method comprising: administering to the subject a composition comprising: the activated chimeric receptor being differentially expressed on injured or diseased cells or tissues (e.g., to a different extent compared to normal cells or tissues); The method is designed to target markers or ligands expressed in As used herein, the terms "express,""expressed," and "expression" are given their ordinary meanings, allowing for the manifestation of information in a gene or polynucleotide sequence. shall refer to the production of proteins by, for example, activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequences. The expression product itself (eg, the resulting protein) may also be referred to as "expressed" by the cell. Expression products can be characterized as intracellular, extracellular or transmembrane. The term "intracellular" shall be given its ordinary meaning and shall refer to the inside of a cell. The term "extracellular" shall be given its ordinary meaning and shall refer to the outside of the cell. The term "transmembrane" shall be given its normal meaning and shall refer to at least a portion of the polypeptide being embedded in a cell membrane. The term "cytoplasm" shall be given its normal meaning and shall refer to being within the cell membrane and outside the nucleus. As used herein, the terms "treat,""treating," and "treatment" in connection with the administration of therapy to a subject shall be given their ordinary meanings and It refers to the beneficial effects obtained from In some embodiments, treatment of a subject with genetically engineered cells described herein achieves one, two, three, four or more of the effects listed below, for example: i) reducing the severity or ameliorating the disease or symptoms associated therewith; (ii) reducing the duration of symptoms associated with the disease; (iii) protecting against progression of the disease or symptoms associated therewith; (iv) regression of associated symptoms; (v) protection against the occurrence or onset of disease-associated symptoms; (vi) protection against recurrence of disease-associated symptoms; (vii) reduction in subject hospitalization; (viii) length of hospitalization. (ix) increasing the survival rate of subjects with the disease; (x) decreasing the number of symptoms associated with the disease; (xi) enhancing, ameliorating, supplementing, supplementing, or Increase. Administration can be by a variety of routes, including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to the affected tissue. obtain. The dose of NK cells can be conveniently determined for a given subject based on the subject's body weight, the type and condition of the disease, and the aggressiveness of the desired treatment, but depending on the embodiment, about 10 ⁵ cells per kg. cells to about 10 ¹² cells per kg (eg, 10 ⁵ to 10 ⁷ , 10 ⁷ to 10 ¹⁰ , 10 ¹⁰ to 10 ¹² and overlapping ranges thereof). In one embodiment, a dose escalation regimen is used. In some embodiments, a range of NK cells (eg, about 1×10 ⁶ cells/kg to about 1×10 ⁸ cells/kg) is administered. Depending on the embodiment, various types of cancer or infectious diseases may be treated. Various embodiments provided herein include treatment or prevention of the following non-limiting examples of cancers: for example, but not limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia ( AML), adrenocortical cancer, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendiceal cancer, central nervous system cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor (e.g., but not limited to, astrocytoma, spinal cord tumor) , brainstem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumor, Burkitt lymphoma, cervical cancer, colon cancer, chronic lymphocytic leukemia (CLL), chronic Myeloid leukemia (CML), chronic myeloproliferative disorder, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cavity cancer, nasopharynx cancer, liver cancer, lung cancer (such as, but not limited to, non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, Uterine and vaginal cancer.

Additionally, various embodiments provided herein include the treatment or prevention of infectious diseases of the following non-limiting examples, including, but not limited to, infections of bacterial origin. This may include, for example, infections with bacteria from one or more of the following genera: Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila. , Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter licobacter), Legionella (Legionella), Leptospira (Leptospira), Listeria ( Listeria), Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella gella), Staphylococcus, Streptococcus ), Treponema, Vibrio and Yersinia and variants or combinations thereof. In some embodiments, methods are provided for treating various fungal infections, and such infections of fungal origin include, for example, Aspergillus sp., Blastomyces sp., Candida sp. , Coccidioides , Cryptococcus , and Histoplasma , and mutants or combinations thereof. In some embodiments, methods of treating various viral infections (e.g., those caused by one or more viruses) are provided, including: adenoviruses, Coxsackie virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, Deborah virus, human herpes virus type 8, HIV, influenza viruses, measles virus, mumps virus, human papilloma virus, parainfluenza virus, poliovirus, rabies virus, respiratory conjugate virus, rubella virus and varicella-zoster virus.

In some embodiments, also provided herein are at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99% (and ranges therein) homologous, and also exhibit one or more of the functions compared to the respective SEQ ID NOs: 1-68; These functions include, but are not limited to: (i) enhanced proliferation, (ii) enhanced activation, and (iii) binding of NK cells with receptors encoded by the nucleic acid and amino chain sequences. (iv) enhanced homing to tumors or sites of infection; (v) decreased off-target cytotoxic effects; (vi) immunostimulatory cytokines and chemokines (e.g., limited (vii) enhancement of the ability to stimulate additional innate and adaptive immune responses, and (viii) combinations thereof.

Additionally, some embodiments provide amino chain sequences corresponding to any of the nucleic acids disclosed herein while accounting for the degeneracy of the nucleic acid code. Additionally, sequences (nucleic acids or amino acids) that differ from those explicitly disclosed herein, but have functional similarity or equivalence, are also contemplated within the scope of this disclosure. The above includes variants, truncations, substitutions or other types of modifications.

According to some embodiments herein, an activated chimeric receptor comprising an extracellular receptor domain comprising a fragment of CD94, NKG2A, or an NKG2C variant, an effector domain comprising a transmembrane region and an intracellular signaling domain. Polynucleotides encoding the body are provided. In some embodiments, the polynucleotide encodes an effector domain that includes CD16. In some embodiments, the polynucleotide encodes an effector domain that includes NCR1. In some embodiments, the polynucleotide encodes an effector domain that includes NCR2. In some embodiments, the polynucleotide encodes an effector domain that includes NCR3. In some embodiments, the polynucleotide encodes an additional effector domain portion that includes 4-1BB. In some embodiments, the polynucleotide encodes a chimeric receptor comprised of a fragment of CD16 and a CD94, NKG2A, or NKG2C variant. In some embodiments, the polynucleotide encodes a chimeric receptor comprised of a fragment of NCR1 and a CD94, NKG2A, or NKG2C variant. In some embodiments, the polynucleotide encodes a chimeric receptor comprised of a fragment of NCR2 and a CD94, NKG2A, or NKG2C variant. In a further embodiment, the polynucleotide encodes a chimeric receptor comprised of a fragment of CD94, NKG2A, or NKG2C variant conjugated to CD16 and optionally 4-1BB. In some embodiments, CD16 is replaced with NCR1, and in some embodiments with NCR2 or even NCR3, depending on the embodiment. In some embodiments, the effector domain further comprises a GS linker, eg, between 4-1BB and one of CD16, NCR1, NCR2 or NCR3.

In some embodiments, the extracellular receptor domain further comprises a hinge region. In some embodiments, the hinge region includes CD8a. However, in additional embodiments, the hinge region includes one or more linkers, which in some embodiments include GS9, CD8a/GS3, truncated CD8a, GS3, and the like. including.

In some embodiments, the extracellular receptor domain further comprises a CD8a signal peptide. In some embodiments, the effector domain includes one or more hemi-ITAM sequences. In some embodiments, the chimeric receptor does not include DNAX activating protein 10 (DAP10). In some embodiments, the chimeric receptor does not include an ITAM motif, but rather utilizes an alternative signaling region (eg, ITSM, hemitum or other costimulatory region).

In some embodiments, the activated chimeric receptors described herein target cells that express the natural ligand for natural killer group 2 member D (NKG2D), resulting in synergistically enhanced NK cell activity. co-expressed with a chimeric receptor that confers cytotoxicity and cytotoxicity. Accordingly, in some embodiments, polynucleotides encoding NKGD chimeric receptors comprising an extracellular receptor domain, a transmembrane region, and an effector domain comprising a peptide that binds to native NKG2D are also provided, wherein The peptide that binds the ligand is a fragment of NKG2D. In some embodiments, the fragment of NKG2D is encoded by a polynucleotide comprising a fragment of the sequence SEQ ID NO:1. In some embodiments, the fragment of NKG2D comprises the sequence of SEQ ID NO: 2, while in further embodiments the fragment encoding NKG2D is codon optimized, eg, comprises the sequence of SEQ ID NO: 3. In some embodiments, the effector domain includes one or more of CD16, NCR1, NCR2, NCR3, 4-1BB, CD28, NKp80, DAP10, CD3ζ, and 2B4. In some embodiments, these effector domains are conjugated to CD8α. In some embodiments, the NKG2D domain is not full-length or wild-type NKG2D, and in some such embodiments, DAP10 is not used in the chimeric receptor construct. In further embodiments, no CD3ζ or ITAM is used. As discussed herein, a combination of transmembrane and intracellular domains is used in some embodiments to result in synergistic interactions between the components of the NKG2D chimeric receptor, resulting in enhanced cytotoxicity. bring about an effect. In some embodiments, linkers, hinges, or other "spacing" elements are provided in the NKG2D chimeric receptor construct. For example, in some embodiments, the effector domain includes a linker. In some embodiments, the polynucleotide includes a GS linker between portions of the NKG2D chimeric receptor construct, e.g., between any of 4-1BB, CD28, CD16, NCR1, NCR3, CD3ζ, DAP10, 2B4, or NKp80. code. In some embodiments, the effector domain of the NKG2D chimeric receptor includes a linker. In some embodiments, the polynucleotide encodes a GS linker between portions of the NKG2D chimeric receptor construct, eg, any of 4-1BB, CD28, CD16, NCR1, NCR3, 2B4 or NKp80. In some embodiments, chimeric receptors that include a hinge region are provided. In some embodiments, the effector domain of the NKG2D chimeric receptor comprises one or more hemi-ITAM sequences. Additionally, any of the chimeric receptors disclosed herein can also be co-expressed with membrane-bound interleukin 15 (mbIL15).

In some embodiments, the provided polynucleotide is mRNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expression of the activated chimeric receptor. As used herein, the terms "nucleic acid," "nucleotide," and "polynucleotide" shall be given their ordinary meanings, including deoxyribonucleotides, deoxyribonucleic acids, ribonucleotides and ribonucleic acids and their polymeric forms. and single-stranded or double-stranded forms. Nucleic acids include naturally occurring nucleic acids (eg, deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”)) and nucleic acid analogs. Nucleic acid analogs include those that are nucleotides that bind to other nucleotides other than naturally occurring phosphodiester bonds, those that contain non-naturally occurring bases, or those that contain bases that are attached via bonds other than phosphodiester bonds. It will be done. As such, nucleic acid analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids. (PNA), locked nucleic acids (LNA) and the like. As used herein, the term "operably linked", e.g. in relation to a regulatory nucleic acid sequence "operably linked" to a heterologous nucleic acid sequence, shall be given its ordinary meaning. , and that this regulatory nucleic acid sequence is placed into a functional relationship with a heterologous nucleic acid sequence. In the context of an IRES, "operably linked" means a functional link between the nucleic acid sequence containing the internal ribosome entry site and the initiation of the heterologous coding sequence in the middle of the mRNA sequence from which the heterologous coding sequence is translated. Refers to connection. As used herein, the term "vector" shall be given its ordinary meaning and is used to introduce a DNA or RNA sequence (e.g., a foreign gene) into a genetically engineered cell. It refers to a medium capable of transforming cells and promoting the expression (eg, transcription and/or translation) of introduced sequences. Vectors include viruses, plasmids, phages, and the like. The term "chimeric receptor" as used herein shall be given its normal meaning and comprises at least two polypeptide domains that are not found together in nature on a single protein; or a cell surface receptor that includes one or more regions or portions derived from different receptors or signaling molecules (eg, another subtype or type of receptor). The term "chimeric receptor complex" as used herein refers to a first polypeptide that may contain at least two polypeptide domains in combination that are not found together in nature on a single protein; The first polypeptide is associated with a second polypeptide (eg, an adapter polypeptide), a signal transduction molecule, or a stimulatory molecule. Additional terminology relating to the production and use of the chimeric receptors disclosed herein will be readily understood by those skilled in the art and may be found in WO 2014/117121 and US Pat. No. 7,994,298. (each of which is incorporated herein by reference in its entirety).

Additionally provided according to some embodiments is a vector comprising a polynucleotide encoding any of the polynucleotides provided herein, the polynucleotide optionally comprising an activated chimera. A vector operably linked to at least one regulatory element for expression of the receptor. In some embodiments, the vector is a retrovirus.

Further provided herein are engineered natural killer cells comprising the polynucleotides, vectors or activated chimeric receptors disclosed herein. In some embodiments, the NK cells are suitable for use in the prophylactic treatment of disease (eg, cancer and/or infectious disease).

In some embodiments, the polynucleotides disclosed herein further encode short hairpin RNAs (shRNAs) that specifically inhibit the transcription or translation of native NKG2A, where the shRNA is directed to the native NKG2A gene. Sense fragments and antisense fragments that contain a nucleotide sequence that hybridizes under stringent conditions and that contain a nucleotide sequence that is substantially identical to a target sequence in the NKG2A gene that is absent from the NKG2A fragment; Sense fragments are separated by loop fragments. In some embodiments, the polynucleotide is coexpressed with an additional construct encoding membrane-bound interleukin 15 (mbIL15). In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expression of an activated chimeric receptor.

Further, in some embodiments herein, vectors are provided that include a polynucleotide of the disclosure operably linked to at least one regulatory element for expression of an activated chimeric receptor. In some embodiments, the vector is a retrovirus.

In some embodiments, a method for treating or preventing cancer or an infectious disease in a mammal in need thereof, the method comprising administering to said mammal a therapeutically effective amount of NK cells. provided that the NK cell expresses an activated chimeric receptor encoded by a polynucleotide according to the present disclosure. In some embodiments, the NK cells are autologous cells isolated from a patient with cancer or an infectious disease. In some embodiments, the NK cells are allogeneic cells isolated from the donor. In some embodiments, the NK cells lack surface expression of native NKG2A.

In some embodiments, there is provided the use of a polynucleotide according to the present disclosure in the manufacture of a medicament for treating or preventing cancer or infectious disease in a mammal in need thereof. In some embodiments, there is provided use of a vector according to the present disclosure in the manufacture of a medicament for enhancing NK cell cytotoxicity in a mammal in need thereof. In some embodiments, vectors are used in the manufacture of a medicament for treating or preventing cancer or infectious disease in a mammal in need thereof.

In some embodiments, there is provided use of isolated genetically modified natural killer cells according to the present disclosure to enhance NK cell cytotoxicity in a mammal in need thereof. In some embodiments, there is also provided the use of isolated genetically modified natural killer cells according to the present disclosure to treat or prevent cancer or infectious disease in a mammal in need thereof.

In some embodiments, a chimeric natural killer group 2 member C (NKG2C) is provided that includes an extracellular receptor domain, a transmembrane domain, and a cytoplasmic domain, where the extracellular receptor domain has SEQ ID NO: 58. include. In some embodiments, chimeric receptors are provided that include an extracellular receptor domain, a transmembrane region, and a cytoplasmic domain of Natural Killer Group 2 Member A (NKG2A). In some embodiments, the transmembrane region comprises a CD8 transmembrane region. In some embodiments, the cytoplasmic domain comprises one or more of a 4-1BB cytoplasmic domain and a CD3ζ cytoplasmic domain.

In some embodiments, polynucleotides encoding chimeric receptors are provided that include an extracellular domain, a transmembrane domain, and a cytoplasmic domain, where the extracellular domain is natural killer group 2 member C (NKG2C) and the sequence Contains number 58.

In some embodiments, polynucleotides are provided that encode chimeric receptors that include an extracellular receptor domain, a transmembrane region, and a cytoplasmic domain of Natural Killer Group 2 Member A (NKG2A). In some embodiments, the transmembrane region comprises a CD8 transmembrane region. In some embodiments, the cytoplasmic domain comprises one or more of a 4-1BB cytoplasmic domain and a CD3ζ cytoplasmic domain.

Methods The following experimental methods and materials were used in the non-limiting experimental examples disclosed below.

Cell Lines and Culture Conditions Human tumor cell lines SKBR3, HT-29, and U2-OS were purchased from the American Type Culture Collection (ATCC; Rockville, MD). Ewing's sarcoma cell lines ES8 and EW8 were derived from St. Obtained from the tissue repository at Jude Research Hospital (Memphis, TN). Cell lines were maintained in RPMI-1640 (ThermoFisher, Waltham, Mass.); the medium was supplemented with 10% fetal bovine serum (FBS; GE Healthcare, Chicago, IL) and antibiotics. Cell lines were supplied with mouse stem cell virus (MSCV) retroviral vectors carrying either green fluorescent protein (GFP) or luciferase (St. Jude Children's Research Hospital's Vector Development and Production Shared Resource). e, Memphis, TN) transduced.

Expansion of Human NK Cells and Selection of NK Cell Subsets Peripheral blood samples were obtained from anonymous waste byproducts of platelet transfusions from healthy adult donors at the National University Hospital Blood Bank, Singapore.

Mononuclear cells were separated by centrifugation on a Lymphoprep density step (Nycomed, Oslo, Norway) and washed twice in RPMI-1640. To expand NK cells, mononuclear cells were co-cultured with the genetically modified K562-mb15-41BBL cell line. Briefly, peripheral blood mononuclear cells (3×10 ⁶ cells) were cultured in SCGM medium (CellGenix, Freiburg, Switzerland) containing 10% FBS and 40 IU/mL human interleukin (IL)-2 (Novartis, Basel, Switzerland). Germany) in 6-well tissue culture plates with 2×10 ⁶ irradiated (100 Gy) K562-mb15-41BBL cells. Fresh tissue medium and IL-2 were added every 2-3 days. After 7 days of co-culture, residual T cells were removed using Dynabeads CD3 (Thermo Fisher) to generate a cell population containing >90% CD56+CD3-NK cells. Prior to experiments, expanded NK cells were maintained in SCGM with FBS, antibiotics, and 400 IU/mL of IL2.

To deplete NKG2A+ NK cells, expanded NK cells were labeled with allophycocyanin (APC) and anti-NKG2A antibodies conjugated to anti-APC microbeads and separated on LD columns (all from Miltenyi Biotech).

DNA plasmid, retrovirus production and NK cell transduction Plasmids encoding all constructs were synthesized by Genescript (Nanjing, China). ARD114 pseudotyped MSCV retrovirus harboring the construct was used to transduce NK cells. Retroviral vector culture supernatants were added to polypropylene tubes coated with RetroNectin (Takara, Otsu, Japan); after centrifugation and removal of the supernatants, expanded NK cells (3 x 10 ^cells ) were added to the tubes. and left at 37°C for 12 hours; fresh virus supernatant was added every 12 hours for a total of 6 times. Cells were then maintained in RPMI with FBS, antibiotics, and 400 IU/ml IL-2 until the time of the experiment.

Flow cytometric detection of activated receptor expression Surface expression of anti-NKG2C was detected using anti-NKG2C-phycoerythrin (PE) antibody (Miltenyi Biotech). NKG2A expression was detected using anti-NKG2A antibodies conjugated to PE or APC (Miltenyi Biotech). Expression of CD94 was detected using anti-CD94-APC (BD Biosciences).

Cytotoxicity Assay GFP/luciferase transduced target cells were suspended in RPMI-1640 with 10% FBS and plated in 96-well flat bottom plates (Costar, Corning, NY). Plates were placed in the incubator for at least 4 hours to allow cell attachment before adding NK cells. Expanded NK cells expressing various receptors or GFP alone, suspended in RPMI-1640 with 10% FBS, were then added at various effector-to-target (E:T) ratios and together with target cells Co-cultured for hours. At the end of culture, the number of viable cells was determined after adding BrightGlo (Promega, Fitchburg, WI) to the wells using a Flx 800 plate reader (BioTek, Winooski, VT).

Example 1 - Activated High Affinity NKG2C Receptor Constructs As disclosed herein, various activated chimeric receptor constructs containing extracellular receptor domains conjugated to various transmembrane domains are prepared. This experiment was performed to evaluate the expression and cytotoxic activity of an activated natural killer group 2 member C (NKG2C) variant engineered for high affinity for HLA-E/peptide complexes. Activated NKG2C receptor constructs were prepared and tested according to the methods and materials described above. Depending on the construct, the methods used can be easily adjusted to accommodate variations required in the production, expression, and testing of the construct.

FIG. 1A shows engineered NKG2C mutants engineered for high affinity for HLA-E/peptide complexes in complex with CD94 and Dap12 dimers (referred to herein as "D12(SIIS)2C94"). (165-168SIIS) is shown. FIG. 1B represents a schematic representation of a construct containing NKG2C (165-168SIIS), Dap12, and CD94 inserted into an MSCV retroviral vector with green fluorescent protein (GFP) after the internal ribosome entry site (IRES).

The ability of NK cells to effectively express this construct was first evaluated. Figure 2 shows robust expression of the high-affinity activated D12 (SIIS) 2C94 receptor complex in purified NKG2C(+)NKG2A(-) NK cells compared to NK cells transduced with vectors containing GFP only. Flow cytometry data shown. Collectively, these data indicate that engineered activated chimeric receptor constructs can be fully expressed on NK cells, according to some embodiments disclosed herein.

In some embodiments, enhanced expression of a construct can be achieved by repeated transduction of a particular construct into NK cells. In some embodiments, the components of the construct may be delivered to the cell in a single vector or alternatively using multiple vectors. Depending on the embodiment, the construct itself may result in enhanced expression; for example, linear or head-to-tail constructs may have enhanced expression due to the reduced degree of intracellular assembly required by multi-subunit constructs. can lead to expression.

NKG2C/CD94 and NKG2A/CD94 bind to non-classical MHC class I molecules HLA-E/peptide complexes in humans. HLA-E has a highly specialized role in cell recognition by NK cells, and HLA-E has a restricted subset of peptides derived from the signal peptide of classical MHC class I molecules, namely HLA-A, Bonds to B, C, and G. Therefore, NK cells can indirectly monitor the expression of classical MHC class I molecules through the interaction of NKG2C/CD94 and NKG2A/CD94 with HLA-E/peptide complexes. To evaluate the efficacy of the activated NKG2C receptor constructs and activated NKG2A receptor constructs described herein, cancer cells were engineered to express HLA-E with an HLA-G signal peptide. Figure 3 depicts the design and production of HLA-E molecules with HLA-G signal peptides. Figure 3A shows the replacement of HLA-E signal peptide (SEQ ID NO: 69) with HLA-G signal peptide (SEQ ID NO: 70) of HLA-E (referred to as GpHLA-E; HLA-E with HLA-G signal peptide). represents. Figure 3B represents flow cytometry data validating exogenous expression of GpHLA-E in solid tumor cell lines HT29, U2OS, ES8, and EW8.

To assess the efficacy of the transduced NK cell population, cytotoxicity assays were performed using cancer cell lines expressing GpHLA-E. Purified NKG2C(+)NKG2A(-) NK cells expressing D12(SIIS)2C94 were significantly more sensitive to U2OS-GpHLA-E, SKBR3-GpHLA-E, and HT29-GpHLA-E cells than control NK cells. High cytotoxicity was demonstrated at all E:T ratios tested (Figure 4). These data demonstrate that not only can NK cells be engineered to express activated chimeric receptor constructs, but also that cells expressing activated chimeric receptors are activated and exhibit enhanced cytotoxicity against target cells. Provide evidence that it can be fully effective.

To confirm the depletion of NKG2A(+) cells, the presence of which could potentially confound the results of these tests and NK cell-based treatments, flow cytometry analysis was performed. After depletion of NKG2A, the cell population was confirmed to consist of NKG2C (+) NKG2A (-) NK cells. Furthermore, cytotoxicity assays of mock-transduced NK cell populations or D12(SIIS)2C94-transduced NKG2C(+)NKG2A(-) NK cells against genetically modified tumor cell lines revealed that the latter NK cell population had an enhanced Cytotoxicity was demonstrated whether or not cells were incubated with anti-NKG2A antibody Z199 (Figure 5).

Example 2 - Activated Chimeric NKG2A/CD94 Receptor Constructs As disclosed herein, various activated chimeric receptor constructs are prepared that include extracellular receptor domains conjugated to various transmembrane domains. This experiment was performed to evaluate the expression and cytotoxic activity of activated natural killer group 2 member A (NKG2A)/CD94 receptor construct. Activated NKG2A/CD94 receptor constructs were prepared and tested according to the methods and materials described above. Depending on the construct, the methods used can be easily adjusted to accommodate variations required in the production, expression, and testing of the construct.

FIG. 6A depicts a schematic diagram showing a complex consisting of natural killer group 2 member A (NKG2A) and a truncated form of CD94, with deletions in the N-terminal portion of the transmembrane and inhibitory cytoplasmic domains. These domains are replaced by the CD8a transmembrane domain and the cytoplasmic domains of 4-1BB (CD137) and CD3ζ. The activated NKG2A receptor and the chimeric CD94 receptor form an activated complex (referred to herein as "2A/94BBz"). Figure 6B depicts a schematic representation of a construct containing a chimeric CD94 receptor and an activated NKG2A receptor inserted into an MSCV retroviral vector with green fluorescent protein (GFP) following an internal ribosome entry site (IRES).

The ability of NK cells to effectively express these constructs was first evaluated. FIG. 7 depicts flow cytometry data demonstrating robust expression of activated NKG2A/CD94-41BB-CD3z receptors in NKG2A-depleted NK cells compared to mock-transduced NK cell populations. Collectively, these data indicate that modified activated chimeric receptor constructs can be fully expressed on NK cells, according to some embodiments disclosed herein.

To assess the efficacy of the transduced NK cell population, cytotoxicity assays were performed using cancer cell lines expressing GpHLA-E. In a 4-hour cytotoxicity assay, expression of activated NKG2A/CD94-41BB-CD3z receptors on expanded NK cells inhibited cytotoxicity toward U2OS-GpHLA-E and HT29-GpHLA-E cells compared to control NK cells. was significantly enhanced compared to These data demonstrate that not only can NK cells be engineered to express activated chimeric receptor constructs, but also that cells expressing activated chimeric receptors are activated and exhibit enhanced cytotoxicity against target cells. Provide evidence that it can be fully effective.

Example 3 - Antitumor Activity of Activated Chimeric Receptor Constructs To demonstrate the ability of activated NK receptor constructs disclosed herein to exert cytotoxic effects on target cells (e.g., tumor cells), colon A xenograft model of rectal adenocarcinoma was used. HT-29 cells are an immortalized colorectal adenocarcinoma cell line. HT-29 cells were transfected with both GpHLA-E and luciferase so that they expressed HLA-E, which is part of the target recognized by the modified activating receptor used in this example. (D12(SIIS) 2C/94 construct modified for high affinity for HLA-E/peptide complexes (165-168SIIS)). Transduced HT-29 cells were injected intraperitoneally into each of 12 NOD-SCIDIL2RG null mice at a dose of 1×10 ⁵ cells/mouse. Thereafter, NK cells (1×10 ⁷ cells) were injected on days 3, 6, 10 and 13. NK cells were transduced with either GFP alone ("control") or the D12(SIIS)2C/94 construct. Mice also received IL-2 (20,000 IU) by intraperitoneal injection three times per week. Ventral and dorsal bioluminescence was measured using a Xenogen Spectrum instrument to assess tumor burden (each symbol is the average of ventral and dorsal readouts).

As can be seen in the left panel of Figure 10 (no NK cells), the average bioluminescence in each mouse increased over time due to increased tumor burden from the growth/proliferation of injected HT-29 cells. Show that there is. The middle panel (mice injected with NK cells expressing GFP) showed that mean bioluminescence was generally increased, but to a lower extent than the 'No NK' group. In stark contrast, the average bioluminescence in D12(SIIS)2C/94 shows a very limited increase even at approximately day 75, when the experiment ended. A single mouse had a moderate increase in average bioluminescence between approximately 40 and 75 days of the experiment. These data indicate that engineered NK cells expressing activated chimeric receptors have significant anti-tumor activity, even when compared to NK cells that themselves have relatively robust anti-tumor effects.

To further examine the effects of the modified activating receptors disclosed herein, survival curves were constructed and survival rates of mice in various groups were determined. Kaplan-Meier survival curves analyzed by log-rank test using the same experimental group as above are shown in FIG. 11. As shown by (dashed line), mice administered with HT-29 cells but not NK cells reached a survival rate of 0% at about day 60. Similarly, mice treated with NK cells expressing GFP (solid gray line) showed a similar survival pattern, reaching 0% survival by approximately day 80. Statistical evaluation of these groups showed no statistical difference between the NK non-administered group and the control group (Kaplan-Meier survival curve, log-rank test, Figure 11). In contrast to the first two groups (similar to the average bioluminescence data above), the D12(SIIS)2C/94 group showed significantly enhanced survival. Not only did 1 in 4 D12(SIIS)2C/94 mice survive for the entire duration of the experiment, but the duration of survival of all mice at D12(SIIS)2C/94 was increased. Statistical evaluation shows that the survival at D12(SIIS)2C/94 was significant compared with the NK non-administration group and the NK cell administration control group (Kaplan-Meier survival curve, log-rank test, P value (represented in Figure 11). Therefore, these data further demonstrate that, even when compared to natural killer cells (which exhibit relatively high cytotoxicity), NK cells engineered to express activated chimeric receptors are surprisingly effective at targeting target cells. It has been shown to have an enhanced cytotoxic effect against In some embodiments, expression of the activated chimeric receptor on NK cells (e.g., using NK cells that have not been modified to express a high affinity domain as disclosed herein) reduction in treatment frequency, reduction in treatment duration, reduction in required dose of NK cells, and overall improvement in outcomes of NK-based cancer immunotherapy regimens (e.g., reduction in tumor burden). reduction and/or survival enhancement).

It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may fall within the scope of one or more of the inventions. Additionally, all other embodiments presented herein use the disclosure herein with respect to any specific feature, aspect, method, property, characteristic, quality, attribute, element, etc. associated with the embodiment. be able to. Accordingly, it should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for each other to form different aspects of the disclosed invention. Therefore, it is intended that the scope of the invention disclosed herein should not be limited by the specific disclosed embodiments described above. Furthermore, while the invention is susceptible to various modifications and alternative forms, specific examples thereof are shown in the drawings and will herein be described in detail. However, the invention is not to be limited to the particular form or method disclosed; on the contrary, the invention covers various described embodiments and all that come within the spirit and scope of the appended claims. It is to be understood that modifications, equivalents, and alternatives of . Any method disclosed herein does not have to be performed in the order listed. Although the methods disclosed herein include certain actions taken by the practitioner, they may also include any third party instructions for those actions, either explicitly or implicitly. For example, an action such as "administering an expanded population of NK cells" includes "directing administration of an expanded population of NK cells." Additionally, where a feature or aspect of the disclosure is described in terms of the Markush Group, those skilled in the art will appreciate that the disclosure is thereby also described in terms of any individual member of the Markush Group or subgroup of members thereof. You will understand that.

The ranges disclosed herein also include any overlaps, subranges, and combinations thereof. Words such as "up to", "at least", "greater than", "less than", "between" and the like include the recited number. Numbers preceded by terms such as "about" or "approximately" include the recited number. For example, "about 10 nanometers" includes "10 nanometers."
Aspects of the present invention include the following.
Item 1
A polynucleotide encoding an activated chimeric receptor that binds to an HLA class I histocompatibility antigen, alpha chain E (HLA-E)/peptide complex, wherein the activated chimeric receptor binds to the HLA class I histocompatibility antigen, alpha chain E (HLA-E)/peptide complex, transmits an activation and/or co-stimulatory signal after binding to the peptide complex, wherein said activated chimeric receptor
a) an extracellular receptor domain comprising an engineered variant of natural killer group 2 member C (NKG2C), comprising:
unmodified NKG2C has low binding affinity for HLA-E/peptide complexes;
the modified NKG2C variant has enhanced binding affinity for the HLA-E/peptide complex compared to native NKG2C, and
an extracellular receptor domain, wherein the modified NKG2C variant comprises the amino acid sequence of SEQ ID NO: 58; or
b) an extracellular receptor domain comprising a fragment of natural killer group 2 member A (NKG2A),
said fragment of NKG2A is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
native NKG2A transmits an inhibitory signal after binding to the HLA-E/peptide complex, and
an extracellular receptor domain in which said fragment of NKG2A transmits activation and/or costimulatory signals after binding to the HLA-E/peptide complex;
Polynucleotides, including.
Section 2
2. The polynucleotide of paragraph 1, wherein the extracellular receptor domain comprises a modified NKG2C variant conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain.
Section 3
2. The polynucleotide of paragraph 1, wherein the extracellular receptor domain comprises a modified NKG2C variant conjugated to a native NKG2C transmembrane region and a native NKG2C intracellular signaling domain.
Section 4
2. The polynucleotide according to item 1, wherein the modified NKG2C variant is encoded by the nucleic acid sequence of SEQ ID NO: 63, or a fragment thereof.
Section 5
2. The polynucleotide of paragraph 1, wherein the polynucleotide further encodes DNAX activating protein 12 (DAP12).
Item 6
2. The polynucleotide of paragraph 1, wherein the polynucleotide further encodes native CD94 or chimeric CD94.
Section 7
The chimeric CD94 is
(a) an extracellular receptor domain comprising a fragment of CD94; and
(b) Effector domain including transmembrane region and intracellular signaling domain
Item 7. The polynucleotide according to item 6, comprising:
Item 8
8. The polynucleotide of paragraph 7, wherein the effector domain comprises one or more of CD16, NCR1, NCR2, NCR3, 4-1BB, NKp80, DAP10, CD3ζ, 2B4, and fragments thereof.
Section 9
8. The polynucleotide of paragraph 7, wherein the chimeric CD94 receptor comprises a fragment of CD94 conjugated to a CD8a transmembrane domain and an effector domain comprising 4-1BB and CD3ζ.
Item 10
10. The polynucleotide of item 9, wherein the chimeric CD94 is encoded by the nucleic acid sequence of SEQ ID NO: 59.
Item 11
10. The polynucleotide according to item 9, wherein the chimeric CD94 comprises the amino acid sequence of SEQ ID NO: 60.
Item 12
2. The polynucleotide of paragraph 1, wherein the activated chimeric receptor comprises a fragment of NKG2A conjugated to a CD8a transmembrane domain and an effector domain comprising 4-1BB and CD3ζ.
Section 13
13. The polynucleotide of paragraph 12, wherein the activated chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO: 61.
Section 14
13. The polynucleotide of paragraph 12, wherein the activated chimeric receptor comprises the amino acid sequence of SEQ ID NO: 62.
Item 15
15. The polynucleotide according to any one of paragraphs 1 to 14, wherein the activated chimeric receptor further comprises a GS linker.
Section 16
15. The polynucleotide according to any one of paragraphs 1 to 14, wherein the chimeric receptor comprises a hinge region.
Section 17
17. The polynucleotide of paragraph 16, wherein the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 5.
Section 18
17. The polynucleotide of paragraph 16, wherein the hinge region is encoded by a fragment of the nucleic acid sequence of SEQ ID NO: 5.
Section 19
17. The polynucleotide according to item 16, wherein the hinge region comprises a glycine-serine repeat motif having the amino acid sequence of SEQ ID NO: 31.
Section 20
17. The polynucleotide according to Item 16, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 32.
Section 21
17. The polynucleotide according to Item 16, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 33.
Section 22
17. The polynucleotide according to paragraph 16, wherein the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 34.
Item 23
17. The polynucleotide according to item 16, wherein the hinge region includes a portion of a β-adrenergic receptor.
Section 24
24. The polynucleotide according to paragraph 23, wherein the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 40.
Section 25
24. The polynucleotide according to paragraph 23, wherein the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 42.
Section 26
15. The polynucleotide according to any one of paragraphs 1 to 14, wherein the extracellular receptor domain further comprises a CD8a signal peptide, and the signal peptide comprises the nucleic acid sequence of SEQ ID NO: 4.
Section 27
15. The polynucleotide of any one of paragraphs 1-14, wherein the effector domain comprises one or more hemi-ITAM sequences.
Section 28
28. The polynucleotide according to item 27, wherein the hemi-ITAM comprises the amino acid sequence of SEQ ID NO: 14.
Section 29
28. The polynucleotide according to item 27, wherein the hemi-ITAM comprises the amino acid sequence of SEQ ID NO: 37.
Section 30
15. The polynucleotide of any one of paragraphs 1-14, wherein the effector domain comprises one or more ITSM sequences.
Section 31
31. The polynucleotide of paragraph 30, wherein the ITSM comprises the amino acid sequence of SEQ ID NO: 15.
Section 32
31. The polynucleotide of paragraph 30, wherein the ITSM comprises the amino acid sequence of SEQ ID NO: 35.
Section 33
15. The polynucleotide of any one of paragraphs 1-14, wherein the NKG2C variant has at least 80% homology to SEQ ID NO: 63.
Section 34
Polynucleotide according to any one of paragraphs 1 to 14, wherein said fragment of NKG2A has at least 80% homology to SEQ ID NO: 65.
Section 35
Polynucleotide according to any one of paragraphs 1 to 14, wherein said fragment of CD94 has at least 80% homology to SEQ ID NO: 67.
Section 36
The polynucleotide comprises: (a) an extracellular receptor domain comprising a peptide that binds to a natural ligand of natural killer group 2 member D (NKG2D); and (b) an effector domain comprising a transmembrane region and an intracellular signaling domain. 15. The polynucleotide according to any one of paragraphs 1 to 14, further encoding a chimeric receptor comprising:
Section 37
the polynucleotide further encodes a short hairpin RNA (shRNA) that specifically inhibits the transcription or translation of native NKG2A,
wherein the shRNA comprises a nucleotide sequence that hybridizes under stringent conditions to the native NKG2A gene, and wherein the shRNA is substantially identical to a target sequence in the NKG2A gene that is absent from the NKG2A fragment. 15. The polynucleotide of any one of paragraphs 1-14, comprising a sense fragment comprising a nucleotide sequence, and an antisense fragment, wherein the sense and antisense fragments are separated by a loop fragment.
Section 38
38. The polynucleotide of any one of paragraphs 1-37, wherein said polynucleotide is co-expressed with an additional construct encoding membrane-bound interleukin 15 (mbIL15).
Section 39
A genetically modified natural killer cell comprising the polynucleotide according to any one of Items 1 to 14.
Section 40
The genetically modified natural killer cell according to item 39, which is an autologous cell isolated from a patient.
Section 41
The genetically modified natural killer cell according to item 39, which is an allogeneic cell isolated from a donor.
Section 42
40. The genetically modified natural killer cell according to item 39, wherein the NK cell lacks surface expression of native NKG2A.
Section 43
The polynucleotide according to any one of Items 1 to 14, and
(i) a chimera comprising (a) an extracellular receptor domain comprising a peptide that binds to the natural ligand of natural killer group 2 member D (NKG2D); and (b) an effector domain comprising a transmembrane region and an intracellular signaling domain. a polynucleotide encoding a receptor;
(ii) a polynucleotide encoding membrane-bound interleukin 15 (mbIL15);
(iii) a polynucleotide encoding a short hairpin RNA (shRNA) that specifically inhibits the transcription or translation of native NKG2A; and
(iv) Any combination of (i), (ii), and (iii)
A genetically modified natural killer cell comprising an additional polynucleotide selected from.
Section 44
A method for enhancing the cytotoxicity of NK cells in a mammal in need thereof, the method comprising administering NK cells to said mammal, said NK cells comprising any one of items 1 to 14. A method of expressing an activated chimeric receptor encoded by a polynucleotide according to item 1.
Section 45
45. The method according to paragraph 44, wherein the NK cells are autologous cells isolated from a patient.
Section 46
45. The method of paragraph 44, wherein the NK cells are allogeneic cells isolated from a donor.
Section 47
45. The method of paragraph 44, wherein said NK cells lack surface expression of native NKG2A.
Section 48
Use of a polynucleotide according to any one of paragraphs 1 to 14 in the manufacture of a medicament for enhancing the cytotoxicity of NK cells in a mammal in need thereof.

Claims (25)

A genetically modified natural killer (NK) cell expressing an activated chimeric receptor that binds to an HLA class I histocompatibility antigen, alpha chain E (HLA-E)/peptide complex, the activated chimeric receptor comprising: , transmits an activation and/or costimulatory signal after binding to said HLA-E/peptide complex, wherein said activated chimeric receptor is
a) an extracellular receptor domain comprising a fragment of natural killer group 2 member A (NKG2A);
wherein said fragment of NKG2A is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
native NKG2A transmits an inhibitory signal after binding to the HLA-E/peptide complex, and said fragment of NKG2A transmits an activating and/or costimulatory signal after binding to the HLA-E/peptide complex. communicate,
said activated chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO: 61, and said extracellular receptor domain is conjugated to an effector domain comprising a CD8a transmembrane domain and 4-1BB and CD3ζ; or b) an extracellular receptor domain comprising an engineered variant of natural killer group 2 member C (NKG2C);
where unmodified NKG2C has low binding affinity for the HLA-E/peptide complex;
the modified NKG2C variant has enhanced binding affinity for the HLA-E/peptide complex compared to native NKG2C, and
the modified NKG2C variant is encoded by the nucleic acid sequence of SEQ ID NO: 63, and the extracellular receptor domain is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
Genetically modified natural killer (NK) cells, including. The genetically modified NK cell of claim 1 , further comprising a polynucleotide encoding DNAX activating protein 12 (DAP12). The genetically modified NK cell according to claim 1 or 2 , further comprising a chimeric CD94 encoded by the nucleic acid sequence of SEQ ID NO: 59 . The genetically modified NK cell comprises (a) an extracellular receptor domain comprising a peptide that binds to a natural ligand of natural killer group 2 member D (NKG2D); and (b) an effector comprising a transmembrane region and an intracellular signaling domain. The genetically modified NK cell according to any one of claims 1 to 3 , further comprising a chimeric receptor comprising a domain. The genetically modified NK cell further comprises a polynucleotide encoding a short hairpin RNA (shRNA) that specifically inhibits transcription or translation of native NKG2A,
wherein the shRNA comprises a nucleotide sequence that hybridizes under stringent conditions to the native NKG2A gene, and wherein the shRNA is substantially identical to a target sequence in the NKG2A gene that is absent from the NKG2A fragment. Genetically modified NK cells according to any one of claims 1 to 4 , comprising a sense fragment and an antisense fragment comprising a nucleotide sequence, wherein the sense and antisense fragments are separated by a loop fragment. The genetically modified NK cell according to any one of claims 1 to 5 , wherein the NK cell additionally expresses membrane-bound interleukin 15 (mbIL15). The genetically modified NK cell according to any one of claims 1 to 6 , which is an allogeneic cell isolated from a donor. The genetically modified NK cell according to any one of claims 1 to 7 , wherein the NK cell lacks surface expression of native NKG2A. A pharmaceutical composition for enhancing NK cell cytotoxicity in a mammal in need of such enhancement, comprising the genetically modified NK cell according to any one of claims 1 to 8 . an activated chimeric receptor that binds to HLA class I histocompatibility antigen, alpha chain E (HLA-E)/peptide complex, wherein the activated chimeric receptor binds to the HLA-E/peptide complex; After binding, transmits an activation and/or costimulatory signal, wherein said activated chimeric receptor
a) an extracellular receptor domain comprising a fragment of natural killer group 2 member A (NKG2A);
wherein said fragment of NKG2A is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
native NKG2A transmits an inhibitory signal after binding to the HLA-E/peptide complex, and said fragment of NKG2A transmits an activating and/or costimulatory signal after binding to the HLA-E/peptide complex. communicate,
said activated chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO: 61, and said extracellular receptor domain is conjugated to an effector domain comprising a CD8a transmembrane domain and 4-1BB and CD3ζ; or b) an extracellular receptor domain comprising an engineered variant of natural killer group 2 member C (NKG2C);
where unmodified NKG2C has low binding affinity for the HLA-E/peptide complex;
the modified NKG2C variant has enhanced binding affinity for the HLA-E/peptide complex compared to native NKG2C, and
the modified NKG2C variant is encoded by the nucleic acid sequence of SEQ ID NO: 63, and the extracellular receptor domain is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
Activated chimeric receptors, including: 11. The activated chimeric receptor of claim 10 , wherein the extracellular receptor domain comprises an engineered NKG2C variant conjugated to a native NKG2C transmembrane region and a native NKG2C intracellular signaling domain. 12. The activated chimeric receptor of claim 11 , wherein the activated receptor further comprises DNAX activating protein 12 (DAP12). Activated chimeric receptor according to any one of claims 10 to 12 , further comprising chimeric CD94 encoded by the nucleic acid sequence of SEQ ID NO: 59 . A polynucleotide encoding an activated chimeric receptor that binds to an HLA class I histocompatibility antigen, alpha chain E (HLA-E)/peptide complex, wherein the activated chimeric receptor binds to the HLA class I histocompatibility antigen, alpha chain E (HLA-E)/peptide complex, transmits an activation and/or co-stimulatory signal after binding to the peptide complex, wherein said activated chimeric receptor
a) an extracellular receptor domain comprising a fragment of natural killer group 2 member A (NKG2A);
wherein said fragment of NKG2A is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
native NKG2A transmits an inhibitory signal after binding to the HLA-E/peptide complex, and said fragment of NKG2A transmits an activating and/or costimulatory signal after binding to the HLA-E/peptide complex. communicate,
said activated chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO: 61, and said extracellular receptor domain is conjugated to an effector domain comprising a CD8a transmembrane domain and 4-1BB and CD3ζ; or b) an extracellular receptor domain comprising an engineered variant of natural killer group 2 member C (NKG2C);
where unmodified NKG2C has low binding affinity for the HLA-E/peptide complex;
the modified NKG2C variant has enhanced binding affinity for the HLA-E/peptide complex compared to native NKG2C ;
the modified NKG2C variant is encoded by the nucleic acid sequence of SEQ ID NO: 63, and the extracellular receptor domain is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
Polynucleotides, including. 15. The polynucleotide of claim 14 , wherein the polynucleotide further encodes DNAX activating protein 12 (DAP12). 16. The polynucleotide of claim 14 or 15 , further encoding a chimeric CD94 encoded by the nucleic acid sequence of SEQ ID NO: 59 . The polynucleotide comprises (a) an extracellular receptor domain comprising a peptide that binds to the natural ligand of natural killer group 2 member D (NKG2D); and (b) an effector domain comprising a transmembrane region and an intracellular signaling domain. 17. A polynucleotide according to any one of claims 14 to 16 , further encoding a chimeric receptor comprising: the polynucleotide further encodes a short hairpin RNA (shRNA) that specifically inhibits the transcription or translation of native NKG2A,
wherein the shRNA comprises a nucleotide sequence that hybridizes under stringent conditions to the native NKG2A gene, and wherein the shRNA is substantially identical to a target sequence in the NKG2A gene that is absent from the NKG2A fragment. Polynucleotide according to any one of claims 14 to 17 , comprising a sense fragment and an antisense fragment comprising a nucleotide sequence, wherein the sense and antisense fragments are separated by a loop fragment. A polynucleotide according to any one of claims 14 to 18 , wherein said polynucleotide is co-expressed with an additional construct encoding membrane-bound interleukin 15 (mbIL15). 20. A vector comprising a polynucleotide according to any one of claims 14 to 19 , said polynucleotide being operably linked to at least one regulatory element for expression of said activated chimeric receptor. There you are, Vector. 21. The vector of claim 20 , wherein the vector is a retroviral vector . A pharmaceutical composition for the treatment of cancer in a subject, the composition comprising genetically modified natural killer (NK) cells comprising an activated chimeric receptor, the activated chimeric receptor comprising:
a) an extracellular receptor domain comprising a fragment of natural killer group 2 member A (NKG2A);
wherein said fragment of NKG2A is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
native NKG2A transmits an inhibitory signal after binding to the HLA-E/peptide complex;
said activated chimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO: 61, and said fragment of NKG2A transmits an activation and/or costimulatory signal after binding to an HLA-E/peptide complex, and said extracellular receptor domain is conjugated to a CD8a transmembrane domain and an effector domain comprising 4-1BB and CD3ζ; or b) an extracellular receptor comprising an engineered variant of natural killer group 2 member C (NKG2C). domain,
where unmodified NKG2C has low binding affinity for the HLA-E/peptide complex;
the modified NKG2C variant has enhanced binding affinity for the HLA-E/peptide complex compared to native NKG2C, and
the modified NKG2C variant is encoded by the nucleic acid sequence of SEQ ID NO: 63, and the extracellular receptor domain is conjugated to an effector domain comprising a transmembrane region and an intracellular signaling domain;
A pharmaceutical composition comprising. 23. The pharmaceutical composition of claim 22 , wherein the NK cells are allogeneic cells isolated from a donor. 24. The pharmaceutical composition of claim 22 or 23 , wherein the NK cells lack surface expression of native NKG2A. The NK cell is
(i) (a) Natural Killer Group 2 Member D (NKG2D)
Extracellular receptor domains containing peptides that bind to natural ligands
in; and
(b) Effects containing transmembrane regions and intracellular signaling domains
a chimeric receptor comprising a vector domain;
(ii) membrane-bound interleukin 15 (mbIL15);
22 further comprising (iii) a short hairpin RNA (shRNA) that specifically inhibits the transcription or translation of native NKG2A; and (iv) a combination of any of (i), (ii), and (iii). The pharmaceutical composition according to any one of -24 .

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